Methods for generating therapeutic delivery platforms

ABSTRACT

Methods for producing engineered exosomes and other vesicle-like biological targets, including allowing a target vesicle-like structure to react and bind with immunomagnetic particles; capturing the immunomagnetic particle/vesicle complex by applying a magnetic field; further engineering the captured vesicles by surface modifying with additional active moieties or internally loading with active agents; and releasing the engineered vesicle-like structures, such as by photolytically cleaving a linkage between the particle and engineered vesicle-like structures, thereby releasing intact vesicle-like structures which can act as delivery vehicles for therapeutic treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of PCT/US2019/057237,filed Oct. 21, 2019, which claims the priority benefit of U.S.Provisional Patent Application Ser. No. 62/748,470, filed Oct. 21, 2018,entitled MICROFLUIDIC ON-DEMAND CAPTURE, LOADING, AND PHOTO-RELEASE OFEXTRACELLULAR VESICLES AND EXOSOMES AS VACCINE DELIVERY PLATFORM, andthe present application claims the priority benefit of U.S. ProvisionalPatent Application Ser. No. 63/148,781, filed Feb. 12, 2021, each ofwhich is incorporated by reference in its entirety herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 2017-67021-26600awarded by the USDA National Institute of Food and Agriculture andGM103638, GM133794, and CA221536 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

The following application contains a sequence listing in computerreadable format (CRF), submitted as a text file in ASCII format entitled“Sequence_Listing,” created on Apr. 19, 2021, as 13 KB. The content ofthe CRF is hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The disclosure generally relates to methods and materials for harvestingintact carriers or delivery vehicles for delivery of bioactivetherapeutics, such as peptides or proteins, nucleotides, and otheractive agents (e.g., chemicals and drugs).

Description of Related Art

Among all deliverable cells and nanoparticles, live-cell derivedextracellular vesicles, especially exosomes in the nano-size range of30˜150 nm, have shown important roles in intercellular communications inrecent decades. The immune cell-derived exosomes have been welldocumented in the regulation of immune stimulation or suppression,driving inflammatory, autoimmune and infectious disease pathology. Theformation of exosomes begins with the creation of endosomes as theintracellular vesicles. Exosomes are differing from othermembrane-derived microvesicles by originating from multivesicular bodies(MVBs) for cellular secretion. Therefore, exosomes contain specificproteins and nucleic acids and represent their parent cell status andfunctions at the time of formation in parent cells. Among many subtypesof exosomes, the immunogenic exosomes with an intrinsic payload of MHCclass I and II molecules and other co-stimulatory molecules are able tomediate immune responses, which opens up opportunities for thedevelopment of novel delivery platforms which can be used for cancervaccines, immunotherapy delivery, and other delivery associated with invivo transportation.

Compared to other nano-sized delivery systems, such as lipid, polymers,gold and silica material, exosomes are living-cell derived, highlybiocompatible nano-carriers with intrinsic payload, and exhibit muchstronger flexibility in loading desired antigens for effective delivery.Exosomes also eliminate allergenic responses without concerns ofcarrying virulent factors and avoid degradation or loss during delivery.However, the development of exosome-based vaccines is hindered bysubstantial technical difficulties in obtaining pure immunogenicexosomes. The diverse subtypes of exosomes could confound theinvestigation on differentiating different cellular messages. On theother hand, molecular engineering of exosomes through either membranesurface or internal loading could provide an untapped source fordeveloping novel antigenic exosomes.

Bioengineered exosomes as emerging delivery vehicles have gainedsubstantial attention in developing a new generation of cancer vaccines,including recent phase-II trial using IFN-DC-derived exosomes loadedwith MHC I/II restricted cancer antigens to promote T cell and naturalkiller (NK) cell-based immune responses in non-small cell lung cancerpatients. Unfortunately, current exosome engineering approaches, such asthe transfection or extrusion of parent cells, and membranepermeabilization of secreted exosomes, suffer from poor yield, lowpurity, and time-consuming operations. There is a need for methods ofproducing exosomes to solve this bottleneck problem. Due to theintrinsic features in automation and high-efficient mass transport,microfluidic systems overcome many drawbacks of benchtop systems andshow superior performance in isolating, detecting and molecularprofiling of exosomes. However, molecular engineering of exosomes usingmicrofluidic platform has not been explored. Presently, the mostreported work on processing exosomes is either in small quality or boundto solid surface/particles, and they are unable to stay intact fordownstream therapeutic preparations.

SUMMARY

In one aspect the present disclosure concerns methods and materials forcapture and photorelease of extracellular vesicles from a biologicalsample. The methods and materials are suitable for use in non-invasiveprocedures which involve testing samples for use in monitoring thetreatment of, and/or diagnosing and/or aiding in the diagnosis, of adisorder or condition that is correlated with the presence of one ormore detectable markers that are contained within extracellular vesiclesthat may be present in the sample. In one or more embodiments, themethods and materials can be used to detect and capture subtypes ofextracellular vesicles. For example, immunomagnetic particles can bedesigned with respective targeting moieties with specificity for asubtype of extracellular vesicle in a population of extracellularvesicles. The population of extracellular vesicles can be sequentiallycontacted with different mixtures of immunomagnetic particles tosequentially sort and/or capture different subtypes of extracellularvesicles. In one example, the population of initially capturedextracellular vesicles can be sequentially contacted with immunomagneticparticles first comprising targeting moieties for CD36, followed bycontacting with immunomagnetic particles comprising targeting moietiesfor CD81, followed by contacting with immunomagnetic particlescomprising targeting moieties for L1CAM. At each stage, the capturedextracellular vesicles can be separated from the rest of the population(e.g., using magnetic immobilization or other filtering technique). Thesorted and or separated extracellular vesicle populations can then beanalyzed separately.

The methods are also suitable for engineering the extracellular vesiclesthemselves by attaching one or more moieties thereto for subsequent invivo administration as a carrier for delivering a therapeutic or activeagent. In embodiments, any biological sample can be used, and can betested directly, or can be subjected to a processing step before beingtested.

In general, the methods comprise contacting a test sample with aplurality of immunomagnetic particles. In one or more embodiments, thesample is a fluid contained within a test container. In one or moreembodiments, the sample may be a solid, which is first dispersed in asuitable buffer system, which is then contained within a test container.Samples may be subjected to pre-processing to either concentrate thesample and/or dilute the sample as desired. In one or more embodiments,the plurality of immunomagnetic particles are added to the testcontainer before, after, or simultaneously with the sample. In one ormore embodiments, the sample and immunomagnetic particles areintermixed, such as via gentle agitation, vibration, shaking, stirring,and the like, to yield a test mixture. In one or more embodiments, themagnetic aspect of the immunomagnetic particles can itself be used tofacilitate mixing of the sample with the particles (e.g., by using anexternal magnetic to swirl the particles throughout the sample). In oneor more embodiments, the test sample and immunomagnetic particles areallowed to mix for a sufficient period of time to ensure complete mixingand reaction with target extracellular vesicles which may be present inthe sample. In one or more embodiments, the sample and particles areallowed to mix for at least 30 minutes. In one or more embodiments, thesample and particles are allowed to mix for at least an hour, at leasttwo hours, at least 3 hours, at least 4 hours, at least 5 hours, atleast 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, atleast 10 hours, at least 11 hours, at least 12 hours (e.g., overnight).In one or more embodiments, the test sample and immunomagnetic particlesare mixed at room temperature or below, preferably at decreasedtemperature (˜4 □C) conditions. It will be appreciated that theparticular incubation conditions and times may depend on and can beoptimized depending upon the selected targeting moiety used and itstarget ligand on the target extracellular vesicles (i.e., the affinitypairs). For example, longer times and/or reduced temperatures canfacilitate binding between certain affinity pairs, whereas faster timesand/or increased temperatures can facilitate binding between otheraffinity pairs.

Once the sample and immunomagnetic particles have sufficiently mixed,and target extracellular vesicles which may be present in the samplehave had adequate opportunity to react with the immunomagneticparticles, an external magnet is applied to the outside of the testcontainer, preferably at a single location (e.g., along an edge sidewallor bottom) to magnetically direct, collect, and immobilize theimmunomagnetic particles in the test mixture at a single location. Inone or more embodiments, the test container is positioned in a holderconfigured to present a magnet below the test container, thereby pullingthe immunomagnetic particles down to the bottom of the container as aconcentrated pellet. The text mixture is then washed 1-3 times using asuitable buffer. Advantageously, using magnetic immobilization meansthat the immunomagnetic particles and bound extracellular vesicles (ifany) will remain immobilized at the location in the test containerduring the washing.

The washed particles and bound extracellular vesicles (if any) are thenresuspended in buffer to create a photorelease mixture. In one or moreembodiments, the photorelease mixture may be transferred to a newcontainer or the photorelease step may be carried out in test container(“photorelease container”). In one or more embodiments, the newcontainer is preferably a clear container, more preferably, a glasscontainer to facilitate transmission of irradiation through thecontainer and into the mixture for the photorelease step. In one or moreembodiments, the photorelease mixture is exposed to activating radiationof the appropriate wavelength depending upon the photocleavable linkerused in the immunomagnetic particles. Exposure times and total exposuredosages can vary depending upon the source of radiation and thephotocleavable linkers.

After photorelease, an external magnet is applied to the outside of thephotorelease container, preferably at a single location (e.g., along anedge sidewall or bottom) to magnetically direct, collect, and immobilizethe immunomagnetic particles in the photorelease mixture at a singlelocation. In one or more embodiments, the photorelease container ispositioned in a holder configured to present a magnet below thephotorelease container, thereby pulling the immunomagnetic particlesdown to the bottom of the container as a concentrated pellet. Thisconcentrated pellet contains the released immunomagnetic particles,whereas the previously-captured extracellular vesicles have been cleavedfrom the particles remain suspended in the supernatant. Thepreviously-captured extracellular vesicles can then be collected andanalyzed. In one or more embodiments, the supernatant containing thepreviously-captured extracellular vesicles is poured or pipetted out ofthe container. The previously-captured extracellular vesicles can thenbe isolated for analysis (e.g., using one or more rounds ofcentrifugation, and the like). An overview of the process is depicted inthe Figures. Suitable containers include any vessel configured to hold aliquid, including, without limitation, microfluidics chambers, testtubes, centrifuge tubes, microtubes, beakers, vials, flasks, bottles,and other glassware, ELISA well plate, microtiter plates, 6-well,12-well, 24-well, 48-well, 96-well, or 384-well plates, cell culturecontainers (petri dishes, bioreactors, 3D culture containers), and otherfluid holding containers.

Various immunomagnetic particles with photocleavable linkers can be usedin the foregoing process, including those described in co-pendingPCT/US2019/057237, filed Oct. 21, 2019, and incorporated by reference inits entirety herein. Particularly preferred particles for use in thesesystems are graphene oxide-based magnetic particles, such as describedin U.S. Pat. No. 10,788,486, filed Oct. 9, 2017, and incorporated byreference in its entirety herein.

Advantageously, this targeted approach, particularly when using grapheneoxide-based immunomagnetic particles, results in highly purifiedpopulations of intact extracellular vesicles or exosomes. It will beappreciated that such pure populations enable more sensitive and/or morespecific detection of target biomarkers, with less background noise formore accurate diagnostics. Such extracellular vesicles, once isolated,can be mined for various diagnostic values, including nucleic acid-basedbiomarkers for various diseases, proteomics, or multi-omics. The drugloading and engineering of extracellular vesicles also can beimplemented in the protocols for developing therapeutics and drugdelivery. The reproducible isolation of specific intact sEVsubpopulations are essential to support well-controlled and precisiondrug delivery in vivo. The prepared target extracellular vesicles canalso be released from the targeting moieties, leaving behind intacttarget extracellular vesicles with potential benefits.

Microfluidic methods and devices are also described herein as oneexemplary, but not limiting, way to capture and/or engineer a variety ofbiologic carriers or delivery vehicles, such as cells, extracellularvesicles and exosomes, and membrane or lipid particles, and polymerparticles. It will be appreciated that the methods can involvemicrofluidics for all or only a portion of the capture, release, and/oranalysis processes. Likewise, as exemplified below, the processes andmaterials are not limited to microfluidics applications.

Regardless of the embodiment, these carriers can be captured using theinventive methods and devices, loaded with active agents (either surfacemodified or encapsulated), and then released as intact, engineeredcarriers for delivering a variety of therapeutic compounds and bioactiveagents. The engineered carriers can be used for diagnostics,prognostics, companion assays, pharmaceutics and therapeutics,immunotherapy and vaccine delivery, and tissue delivery, and other usageassociated with in vivo transportation of active agents. Embodimentsdescribed herein are exemplified with respect to exosomes. However, itwill be appreciated that exosomes represent a particularly challengingbiological target, such that it is envisioned that the platform can beapplied to other similar structures—vesicular or vesicle-like structurescharacterized by a liquid core and membrane or bilayer—including cells(including T-cells), microsomes, and the like. These biological targets,which are then engineered into carriers or delivery vehicles can also becharacterized as nanocarriers.

In one exemplary aspect, the present disclosure concerns microfluidicanalytical devices and method of on-demand capture, loading, andphoto-release of intact engineered nanocarriers. The microfluidicdevices can enable real-time harvesting and antigenic modification ofextracellular vesicles, particularly exosomes, with subsequent releaseof intact exosomes downstream on-demand.

Also disclosed for use in any of the described embodiments aremagnetic-nanoparticles functionalized with photo-cleavable, affinityprobes (active moieties) for capturing and on-demand releasing MHC-Ipositive exosomes via a light trigger. The affinity probe can include anantigenic peptide, antibody, aptamer, nanobody and other affinity-basedprobes. The photo-release of the modified/loaded exosomes in themicrofluidic devices or other suitable vessel can be well controlledspatially and temporally with 95% or greater efficiency. Such a culturesystem allows antigenic engineering of exosomes either through mediatingtheir parent cell growth using stimulations, or direct molecularengineering on the surface of produced exosomes. Heterogeneity ofexosome subtypes has been found from the same population of parentcells. The released subtypes of exosomes contain distinct molecular andbiological properties for different cellular regulation. The disclosedmethods can capture, load, and release specific subtype of exosomes withmore targeted therapeutic functions. The carriers that can be used inthis disclosed method for capture, loading and release include cells,extracellular vesicles and exosomes, and membrane or lipid particles,and polymer particles, which can encapsulate drugs (small moleculecompounds), genes and bioactive therapeutics. Proof of concept withseveral tumor antigenic peptides (e.g., gp-100, MAGE-A3, and MART-1)which are commonly used in developing cancer vaccines but difficult indelivery due to the degradation have been demonstrated herein. Themicrofluidic devices are used by way of example to show high-efficiencyin engineering immunogenic exosomes (MHC I+), meanwhile, photo-releasingthe intact functional exosomes downstream. Cellular uptake of engineeredexosomes by antigen presentation cells has also been demonstrated, whichdisplayed much-improved internalization ability compared tonon-engineered exosomes. In particular, the engineered exosomes showsignificantly higher activation rate (at least 30%) for activating Tcells by challenging CD8 T cells purified from the spleen of 2 Pmel1transgenic mice, than non-engineered exosomes. We also assessed thedegree of potency of antimicrobial peptide-engineered immunogenicexosomes for stimulating T cells ex vivo using transgenic mice fortreating bovine respiratory syncytial virus (BRSV) infections. Exosomesengineered with BRSV targeting peptide (Peptide 4: M187-195 peptideNAITNAKII, SEQ ID NO:4) have the capacity to activate BRSV M-specific Tcells in the presence of activated dendritic cells.

An important aspect of the methods and materials is that they are ableto capture and release for analysis or use intact extracellularvesicles, meaning that their membranes remain intact and unbroken orundamaged (i.e., the vesicle has not ruptured) and have no relevantcomponent removed or destroyed by the capture and release process.Accordingly, the engineered extracellular vesicles are viable andfunctional for application in cancer immunotherapy and vaccination forinfectious disease, among other uses. This approach for producingengineered vesicles not only provides an enabling strategy forhigh-efficiency production of purified, enriched therapeutic vesiclesbut also serve as an investigation tool for understanding roles ofvariable peptide-engineered exosomes in antitumor immune responses,cancer immunotherapy, and vaccination for treating infections.

In specific aspects, the microfluidic devices disclosed herein cancomprise a cell culture chamber dimensioned to maintain biologicalmaterial in a three-dimensional configuration; a mixing channel fluidlyconnected to the cell culture chamber and comprising a plurality ofsample inlet channels disposed along the mixing channel, wherein theratio of a width of the cell culture chamber to the largestcross-sectional dimension of the mixing channel is at least 5:1; anisolation channel defining a path for fluid flow from the mixing channelto an isolation outlet; and a collection chamber fluidly connected tothe isolation outlet and comprises a magnet operatively coupled to thecollection chamber to produce a magnetic field within the collectionchamber.

In other aspects, the microfluidic devices can comprise a cell culturechamber comprising a cell culture inlet and a cell culture outlet; afluid inlet channel and a particle inlet channel, wherein the cellculture outlet, the fluid inlet channel, and the particle inlet channelfluidly converge at a mixing intersection; a mixing channel fluidlyconnected to the mixing intersection and defining a path for fluid flowfrom the mixing intersection to a mixing outlet, wherein the ratio of awidth of the cell culture chamber to the largest cross-sectionaldimension of the mixing channel is at least 5:1; and a collectionchamber fluidly connected to the mixing outlet and comprises a magnetoperatively coupled to the collection chamber to produce a magneticfield within the collection chamber. In these aspects, the mixingchannel can comprise an isolation channel disposed between the mixingintersection and the mixing outlet.

The isolation channel in the microfluidic devices can have a geometry toinduces turbulent flow so as to mix flowing fluids in the device. Forexample, the isolation channel in the microfluidic devices can have aserpentine geometry. The isolation channel can further include one ormore channel constriction domain that decreases in width for producing alocal vortex flow profile. In certain embodiments, the isolation channelcan comprise a plurality of channel constriction domains, preferably atleast 5 channel constriction domains.

As described herein, the microfluidic devices comprise a cell chamberand a mixing channel. The cell chamber and the mixing channel can have aheight and a width. The height and the width of the mixing channel caneach be at least 50 microns, preferably between 50 and 500 microns. Theratio of the cell culture chamber width to the largest cross-sectionaldimension of the mixing channel can be from 5:1 to 500:1, from 5:1 to200:1, from 5:1 to 100:1, from 5:1 to 20:1, preferably from 6:1 to 12:1.The cell culture chamber can have a volume of about 200 microliters orgreater, preferably from about 200 microliters to about 1 milliliter.

The microfluidic devices disclosed herein can further comprise a pumpoperably coupled to the device.

It also disclosed the method of using the device for capture of a targetin a sample solution, loading, and on-demand photo release can be usedfor harvesting intact delivery carriers. Such carriers can be cells,extracellular vesicles and exosomes, and membrane or lipid particles,and polymer particles, which can encapsulate drugs, genes and bioactivetherapeutics. Specifically, this integrated and continuous method ofcapture, loading and photo-release can produce the engineered exosomesin a microfluidic device. The methods comprise introducing a biologicalsample containing exosomes (or another target) into a mixing channel andmixing the exosomes with immunomagnetic particles and a wash buffer toform a mixture; allowing the exosomes to react and affinity bind withthe immunomagnetic particles; and collecting the exosomes bound to theimmunomagnetic particles by applying a magnetic field within acollection chamber. The method for producing the engineered exosomes caninclude introducing cells into a cell culture chamber of themicrofluidic device and first culturing the cells under conditionsallowing the release of exosomes. This can be carried out in-line, inthe same microfluidic device used for capture and loading.

The cells from which the exosomes are released can be selected fromdendritic cells, stem cells, immune cells, megakaryocyte progenitorcells, macrophages, or other live cells.

The immunomagnetic particles bound to the exosomes can comprise amagnetic particle-bound to an affinity probe for capturing exosomes viaa moiety comprising a photocleavable linker. As described herein, theaffinity probe for capturing exosomes can include an antigen peptide,antibody, aptamer, or antigenic epitope thereof for capturing theexosomes. Suitable examples of antigen peptides include MAGE-A3, gp-100,HER-2, p53, PSA-1, or MART-1. The moiety comprising the photocleavablelinker can include biotin bound to immunomagnetic particle and attachedat the other end via a photocleavable linker to the affinity probe. Theaffinity probe targets surface proteins on the target (e.g.,immunostimulatory molecules or markers). Suitable immunostimulatorymolecules include an MHC class I molecule, an MHC class II molecule, aninterleukin, TNFα, IFNγ, RANTES, G-CSF, M-CSF, IFNα, CTAPIII, ENA-78,GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1α, MIP-1β and combinationsthereof. Preferably, the affinity probe is itself an antigenic moiety(peptide) which preferentially binds to a surface protein on the target.

After mixing, the immunomagnetic particles capture/bind exosomes (orother target) in the sample. The immunomagnetic particles areimmobilized in the device, e.g., using a magnet positioned adjacent to acollection chamber. The immobilized bead/exosome complexes are thenwashed and incubated with buffer solution containing active moieties foreither surface loading onto the captured exosomes or internalencapsulation, as described in more detail below. The methods canfurther comprise photolytically cleaving the captured, modified exosomesfrom the immunomagnetic particles, releasing intact, engineered exosomescomprising the active agent (antigen peptide or antigenic epitopethereof).

The methods for producing the engineered biological targets, such asexosomes, can be performed using the microfluidic devices disclosedherein, although the process is not limited to microfluidics and can becarried out in any suitable vessel or series of vessels. In someembodiments, the methods are carried out in real-time. In one or moreembodiments, the methods are streamlined (aka “continuous”), such thatthe capture, loading, and release of the biological target occurs in thesame device/container (i.e., without having to transfer or move betweencontainers or reaction tubes, but rather in-line along a microfluidicchannel and in-line capture/engineering chamber). Thus, each of thesteps can be carried out consecutively, one after the other using thevarious inlets which converge at a single microfluidic channel, andpreferably substantially immediately one after the other. In otherwords, the method involves immunomagnetic bead loading, followedimmediately or nearly simultaneously with sample loading, followed byloading of the active agents to be attached to or loaded into thecaptured target. Each component loaded into the microfluidic deviceconverges from respective inlets into a single microfluidic channel,followed by “automatic” retention in the capture/engineering chamberdownstream from the inlet by the magnet positioned adjacent to thechamber. As the fluid mixture flows through the channel and then thechamber, the respective reactions are occurring in real-time (i.e.,capture and loading). Application of light to the chamber can thenrelease the engineered target. It will be appreciated that thestreamlined process is much faster than traditional benchtop methods.Preferably, the process from sample/bead loading at respective inlets tocollection of the released at the outlet, engineered target can becompleted within approximately 2 hours total, more preferably withinapproximately 90 minutes, and more preferably within approximately 1hour.

Compositions comprising engineered exosomes, particularly immunogenicexosome complexes are also disclosed. The immunogenic exosome complexcan comprise an antigenic peptide or antigenic epitope thereofconjugated to a surface of an exosome, wherein the immunogenic exosomecomplex activates T-cell by at least 30% compared to a native exosome.The antigenic loading of such antigenic peptide or antigenic epitopescan be performed after mixing and capture exosomes for completelycoating exosome surface with antigenic peptides. The compositions can beprepared using the methods disclosed herein. Accordingly, immunogenicexosome complex prepared by a process comprising introducing cells intoa cell culture chamber of a microfluidic device; culturing the cellsunder conditions allowing release of engineered exosomes; introducingthe engineered exosomes into a mixing channel and mixing the engineeredexosomes with immunomagnetic particles and a wash buffer to form amixture for exosome capture/binding; then apply a magnetic field withina collection chamber to collect the isolated exosomes, allowing to reactwith the loading buffer containing antigenic peptides to form animmunogenic exosome complex; and apply UV light to break thephoto-cleavable linker for collecting the immunogenic exosome complex atthe outlet of microfluidic device.

Pharmaceutical compositions comprising the immunogenic exosome complexare also disclosed. The loading targets can be drugs, genes andbioactive therapeutics. Methods of treating disease in a subjectcomprising administering to the subject a pharmaceutical compositioncomprising an immunogenic exosome complex are disclosed. In certainembodiments, the disease can be an infection. In some examples, thedisease can be cancer. When the disease to be treated is cancer, themethods can further include administering a chemotherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates (A) immunomagnetic beads mixed with a samplesolution, (B) capture of targets in a sample solution usingimmunomagnetic beads; and (C) loading of antigenic or active agents ontoor inside of the captured target.

FIG. 2A illustrates (A) application of light or activating radiation tothe bead/target complex, and (B) photorelease of an engineered target(carried) surface modified with active agents or antigenic moieties.

FIG. 2B illustrates (A) application of light or activating radiation tothe bead/target complex, and (B) photorelease of an engineered target(carrier) loaded with active agents inside.

FIG. 3 is an illustration of a process overview for a 3D-printed moldedPDMS microfluidic culture chip for streamlined engineering of antigenicexosomes employed in activating anti-tumor responses.

FIG. 4 illustrates an embodiment of a microfluidic device.

FIG. 5 shows images of (a) a microfluidic channel and flow; (b) mixingwith microbeads; (d) morphology of cells; and (e) SEM image of releasedexosomes.

FIG. 6 illustrates an embodiment of a microfluidic device.

FIG. 7A shows an illustration of immunomagnetic capture and on-demandphoto-release of MHC-I positive, immunogenic exosomes.

FIG. 7B shows characterization of three tumor-targeting peptide antigensconjugated with photo-cleavable immunomagnetic beads for binding andphoto-release of fluorescence-labeled immunogenic exosomes. The MHC-Iantibody is used as the positive control to compare the binding strengthbetween MHC-I positive exosomes and tumor targeting peptides.

FIG. 8A show characterization of the performance of on-demandphoto-release of captured exosomes from immunomagnetic capture beads.The positive control is a fluorescence-labeled antibody captured byphoto-release immunomagnetic beads. The negative control is theimmunomagnetic beads without a photo-cleavable linker.

FIG. 8B shows the SEM image of a surface of photo-release immunomagneticbeads captured with exosomes. Exosome particles were seen as the cupshape due to the vacuum sample preparation.

FIG. 8C shows the SEM image of the surface of photo-releaseimmunomagnetic beads after photocleavage.

FIG. 8D shows characterization of UV exposure time influence on thephoto-cleavage efficiency.

FIG. 8E shows nanoparticle tracking analysis of exosome sizedistribution between engineered exosomes and non-engineered exosomes.

FIG. 9A shows the confocal microscope images of DC uptake of tumortargeting antigenic (TTA) peptide, gp-100 surface engineered exosomes,compared with non-engineered exosomes. The image was taken every hourfor tracking the green fluorescence labeled exosomes uptake by DCs (cellnuclei were stained with DAPI).

FIG. 9B shows the release of cytokine IFN-γ from DCs culture measured byELISA for monitoring 48 hours, compared between non-engineered exosomesand gp-100 engineered exosomes.

FIG. 10A depicts representative flow plots from wells containing Tcells+activated JAWS cells with increasing concentrations of thegp100-engineered exosomes.

FIG. 10B depicts the cumulative data from all three culture conditionsshowing the CD8+ T cell dividing rate under stimulation. The results arerepresentative of 2 independent experiments with duplicate wells foreach culture condition.

FIG. 11 depicts flow cytometry plots from wells containing T cells andactivated JAWS cells with increasing concentrations of the bovinerespiratory syncytial virus (BRSV) antimicrobial peptide-engineeredexosomes for depicting the immunogenic potency.

FIG. 12A illustrates a 3D printing approach for producing 3D moldintegrated with cell culture and downstream exosome isolation, surfaceengineering, and on-demand photo release.

FIG. 12B shows the results from replicating PDMS microfluidic device.

FIG. 13 shows results from investigation of the side-effect of UVexposure on exosome molecular contents in terms of proteins, DNAs andRNAs.

FIG. 14 shows dendritic monocytes culture under different stimulationconditions: dendritic monocytes without any stimulation (negativecontrol; first image); PWM protein stimulation (positive control; secondimage); and gp-100 engineered exosome stimulation (last image).

FIG. 15A is a schematic illustration of the fabrication of Nano Pom Pomsimmunomagnetic particles.

FIG. 15B shows TEM (top) and SEM (bottom) images showing the unique 3Dnano-scale pom poms-like morphology from fabricated NanoPomsimmunomagnetic particles (right panel) compared to commercialimmunomagnetic beads (left panel).

FIG. 15C shows TEM imaging of the surface of NanoPoms captured with sEVsvalidated by antiCD63 gold nanoparticle immunestaining. The insert showsthe captured single EV in the size range of ˜100 nm with three goldnanoparticles bound (˜10 nm).

FIG. 15D is a graph of the NTA analysis of NanoPoms isolated sEVs incomparison with UC isolated EVs.

FIG. 15E shows SEM imaging of captured sEVs which are completelycovering NanoPoms immunomagnetic particles, and are completely releasedfrom NanoPoms particles after photorelease. The scale bar is 100 nm. Thephotoreleased sEVs can be collected on-demand for downstreamapplications including DNA NGS, small RNA NGS, western blotting andproteomic analysis, as well as the in vivo delivery.

FIG. 16A is a graph from X-Ray Photoelectron Spectroscopy (XPS) analysisof NanoPoms immunomagnetic particle surface properties withextracellular vesicles captured. The immunomagnetic particles withoutthe 3D-structured nanographene serves as the negative control. Thecommercial dynabeads were used as the positive control.

FIG. 16B shows SEM images showing the surface morphology of NanoPomsparticles before EV capture, after EV capture, and after release ofcaptured EVs. The scale bar is 100 nm. The dense round small particleswere seen covering NanoPoms particle surface completely after captureand can be completely release from particles for harvesting intact sEVs.

FIG. 16C shows fluorescence microscopic images showing the NanoPomsparticles bound to FITC-biotin after conjugation with streptavidin, withdyna streptavidin beads as the positive control, which exhibits the muchbrighter fluorescence from NanoPoms particles indicating more bindingsites.

FIG. 17 shows SEM and TEM images (top row) showing the morphology ofNanoPoms particle captured sEVs from a variety of biological fluids:sEVs captured from ovarian cancer patient plasma (left), sEVs capturedfrom the cow's milk with enlarged insert showing the classic cup shape(middle), sEVs captured and released from the Wharton's jellymesenchymal stem cell culture medium (right). The scale bar indicatesthe 100 nm; and TEM images (bottom row) showing the much clean anduniform sEVs prepared from NanoPoms isolation from cell culture medium.In contrast, ultracentrifugation isolation prepares EVs in a mixturewith small aggregates and debris.

FIG. 18 shows a graph and corresponding photographic image from theoptimization of NanoPoms particles concentration used for isolating sEVsfrom 1 mL milk solution, characterized by the Pierce BCA Protein Assay.The five repetitive measurements were performed for each data point withRSD<˜5% (n=5).

FIG. 19A shows results from DNA NGS analysis of 11 BC patient urinesamples with 4 healthy individuals as the control group using GeneReadAIT panel. The EVs were prepared in parallel by UC, NanoPoms, andcontrol bead approaches to extract total DNAs shown in the bar graphs.The most frequent 1,411 cancer relevant variants were sequenced.

FIG. 19B shows the results from NGS GeneRead analysis of tumor cell DNAsfrom the matched BC patient, compared with urinary EV DNAs prepared byUC, NanoPoms, and commercial beads.

FIG. 19C shows the digital droplet PCR analysis of EGFR (Thr790Met)extracted from purified EVs using both NanoPoms (pink dots) and UC (bluedots) approaches from 30 bladder cancer urine samples with 10 healthyindividuals as the control group. d) Receiver operating characteristic(ROC) analysis of ddPCR detection of EGFR showing diagnostic performanceusing NanoPoms prepared DNAs compared with UC preparation. The a.u.c(Area Under the Curve) for NanoPoms preparation is 0.78 with p=0.01. Thea.u.c. for UC preparation is 0.71 with p=0.04.

FIG. 20 is a graph of the ddPCR analysis of the mutation frequency usingDNAs isolated from either NanoPoms or ultracentrifugation (UC) preparedurinary EVs.

FIG. 21 shows the ddPCR analysis of NanoPoms isolated sEV DNAs showedthe EGFR heterozygosity in the three bladder cancer patients. Incontrast, UC isolated EV DNAs from the same sample input did not detectthe EGFR heterozygosity in patient 2 and patient 3. The DNA copy numberwas substantially lower in UC prepared EV DNAs as compared to NanoPoms.

FIG. 22A shows the ddPCR analysis of DNAs from NanoPoms prepared urinarysEVs for detecting EGFR (Thr790Met) in three BC patients, compared withUC EV preparation.

FIG. 22B shows the Sanger sequence validation of NanoPoms preparedplasma sEV EGFR heterozygosity from matched patients in FIG. 22A. DNAsfrom the corresponding patients' white blood cells (WBC) as thewild-type control.

FIG. 23A shows the distribution of small RNA categories from bothNanoPoms and UC prepared urinary EVs from BC patient and healthycontrol.

FIG. 23B is a heatmap with dendrogram clustering analysis depicts thetop 100 highly expressed miRNAs from urinary EVs isolated from both BCpatient and healthy individual using UC, NanoPoms without or withoutlight release process. Red color indicates a higher expression z-score.Hierarchical clustering was performed, using the Spearman correlationmethod. NanoPoms isolation approach with or without light releaseprocesses have been clustered together due to higher similarities intheir transcript expressions.

FIG. 23C is a Volcano plot analysis that depicts the most biologicallysignificant urinary EV miRANs with large fold changes identified byusing NanoPoms preparation compared to UC preparation. Top 5 highlysignificant miRNAs are labeled in plot, which are from NanoPomspreparation.

FIG. 24 is a heatmap with dendrogram clustering analysis depicts the top100 highly expressed miRNAs from urinary EVs derived both the BC patientand the healthy control which were isolated by the NanoPoms approachwith and without light release process. Red color indicates a higherexpression z-score. Hierarchical clustering was performed, using theSpearman correlation method.

FIG. 25 A shows Western blotting analysis of urinary EV proteinsprepared by both NanoPoms and UC approaches. Two BC patients and onehealthy individual urine samples were used with HTB9 cells and their EVsfrom conditioned media as the control. Protein loading amount isconsistent between samples (˜5 μg).

FIG. 25C shows a Venn diagram illustrating the relationship of proteomesfrom BC and healthy urinary EVs prepared by the NanoPoms approach, withreferences from ExoCarta Exosome Protein Database and the UrinaryExosome Protein Database.

FIG. 25C shows Gene Ontology enrichment analysis of differentlyexpressed proteins from NanoPoms prepared BC urinary sEVs. Most abundantitems are listed in biological process, cell component and molecularfunction, respectively.

FIG. 26A shows representative IVIS images at 24 hours, 48 hours, and 72hours post-injection of live mice. The HTB9 tumor cell derived sEVs andnon-malignant HEK cell derived sEVs with DiR labeling (2.0×109particles/ml) were prepared by NanoPoms approach for intravenous tailinjection into BALB/cJ mice. The buffer solution without EVs was used asthe negative control.

FIG. 26B shows representative IVIS images of harvested organs (lung,liver, kidney, spleen, heart, and brain) at 48 hours and 72 hours postinjection from mice.

FIG. 26C shows the fluorescence signals normalized with negative controlfrom IVIS images in each organ harvested at 48 hours and 72 hours postinjection (n=2, mean±SD).

DETAILED DESCRIPTION

The following description of the disclosure is provided as an enablingteaching of the disclosure in its best, currently known embodiment(s).To this end, those skilled in the relevant art will recognize andappreciate that many changes can be made to the various embodiments ofthe invention described herein, while still obtaining the beneficialresults of the present disclosure. It will also be apparent that some ofthe desired benefits of the present disclosure can be obtained byselecting some of the features of the present disclosure withoututilizing other features. Accordingly, those who work in the art willrecognize that many modifications and adaptations to the presentdisclosure are possible and can even be desirable in certaincircumstances and are a part of the present disclosure. Thus, thefollowing description is provided as illustrative of the principles ofthe present disclosure and not in limitation thereof.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. The following definitions areprovided for the full understanding of terms used in this specification.

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular fluidic channel is disclosed and discussed anda number of modifications that can be made to the fluidic channels arediscussed, specifically contemplated is each and every combination andpermutation of the fluidic channels and the modifications that arepossible unless specifically indicated to the contrary. Thus, if a classof fluidic channels A, B, and C are disclosed as well as a class offluidic channels D, E, and F and an example of a combination fluidicchannels, or, for example, a combination fluidic channels comprising A-Dis disclosed, then even if each is not individually recited each isindividually and collectively contemplated meaning combinations, A-E,A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed.Likewise, any subset or combination of these is also disclosed. Thus,for example, the sub-group of A-E, B-F, and C-E would be considereddisclosed. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods.

It is understood that the devices disclosed herein have certainfunctions. Disclosed are certain structural requirements for performingthe disclosed functions, and it is understood that there are a varietyof structures which can perform the same function which are related tothe disclosed structures, and that these structures will ultimatelyachieve the same result.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Thus, where a method claim does notexpressly recite an order of steps to be followed or it is not otherwisespecifically stated in the claims or description that the steps are tobe limited to a specific order, it is no way intended that an order beinferred, in any respect. This holds for any possible non-express basisfor interpretation, including: matters of logic with respect toarrangement of steps or operational flow; plain meaning derived fromgrammatical organization or punctuation; and the number or type ofembodiments described in the specification.

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an agent” includes a plurality ofagents, including mixtures thereof.

As used herein, the terms “can,” “may,” “optionally,” “can optionally,”and “may optionally” are used interchangeably and are meant to includecases in which the condition occurs as well as cases in which thecondition does not occur. Thus, for example, the statement that aformulation “may include an excipient” is meant to include cases inwhich the formulation includes an excipient as well as cases in whichthe formulation does not include an excipient.

Ranges can be expressed as from “about” one particular value, and/or to“about” another particular value. When such a range is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. It is alsounderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed.

The terms “upstream” and “downstream” refer to positions within a devicewhich are relative another position and a direction of fluid flow. Asused herein, the term “upstream” refers to a first position that islocated in a direction opposite the direction of fluid flow relative toa second position. Conversely, as used herein, the term “downstream”refers to a second position that is located in a direction along thedirection of fluid flow relative to a first position.

Methods

With reference to FIG. 1, the general methods described herein involve aplurality of immunomagnetic particles (beads), which are mixed with asample solution suspected of containing a target population ofextracellular vesicles. In some embodiments, a biological sample iscollected from a subject and prepared for the method, e.g., by dilutingwith buffer, concentrating, etc. In some embodiments, cells arecollected and expanded in culture. In some embodiments, cells arecollected and cultured under conditions to release exosomes or otherextracellular vesicles or vesicle-like structures into culture. In anyevent, the immunomagnetic particles contain a photocleavable linker andaffinity probe (and preferably a plurality of photocleavable linkers,each with a respective affinity probe) extending from the particlesurface for capturing the target. The immunomagnetic particles arecontacted with a sample solution for a period of time sufficient for thetarget (if present in the sample) to interact with the affinity probesextending from the immunomagnetic particles. In FIG. 1, a single bead isdepicted with a single linker for ease of illustration; however, inpractice, each immunomagnetic particle will be coated with a pluralityof linkers (preferably substantially the entire surface area of theparticle/bead is coated with linkers). Moreover, the relative sizes inFIG. 1 are not to scale, but enlarged for illustration purposes. Inpractice, the bead/particle is preferably at least 5 times larger thanthe target (e.g., in the case of exosomes, which range 30-150 nm indiameter, the bead is preferably 500 nm or larger, preferably from about500 nm to about 1000 nm or from about 500 nm to about 800 nm, but beadsas small as 5 nm to about 100 nm can be used in some embodiments). Inthis way, a plurality of targets (e.g., extracellular vesicles) will becaptured on the surface of a single bead/particle. FIG. 1 uses a singlebead and target interaction for ease of reference. As shown in FIG.1(A), the bead and sample solution are mixed for a sufficient period oftime (e.g., in a mixing chamber or channel in the microfluidic device).If the target is present in the sample solution, it will be captured bythe bead (and specifically by an affinity probe extending from the beadvia its photocleavable linker), as illustrated in FIG. 1(B). Exemplaryphotocleavable linkers are described herein, and may include linearchains including biotin or similar moiety at one end for attachment tothe particle and an amine moiety at the other end for attachment to theaffinity probe. A preferred linker has the following structure, wherethe dashed line indicates the bond cleaved during photo exposure, andthe “binding group” represents the moiety (e.g., biotin) used to attachthe linker to the bead (directly or via a functionalized surfacecoating, e.g., avidin):

Although various embodiments are described herein, the affinity “probe”is typically an oligopeptide sequence that has specificity for andrecognizes the target, such as a peptide that recognizes and acts as areceptor for a surface protein on the target. As used here, the phrase“specificity for” is intended to differentiate the affinity probe fromnon-specific binding or reactions between molecules, and means that theset of specific targets for which the affinity probe can interact islimited, and in some cases even exclusive, such that binding does notoccurs at an appreciable rate with any other molecule except for thetarget (and specifically, its designated surface protein(s)). Shortoligopeptide sequences are preferably used for the affinity probeincluding sequence segments with high specificity for the target. Morepreferably, upon binding, the affinity probe and surface protein createa complex that enhances the immunogenic potential of the target, asdescribed in more detail herein and demonstrated in the workingexamples.

The bead with the captured target (e.g., extracellular vesicles) isimmobilized in the vessel at a first location. The bead can beimmobilized before or after capture of the target. As describedelsewhere herein, this can be achieved by positioning a magnet adjacenta collection or engineering chamber in the microfluidic device or othervessel. As the sample solution and bead solution flow through themicrofluidic channel or are mixed within the vessel, the beads andtarget interact and come into proximity or contact with one anotherthereby capturing the target. The magnetic beads are immobilized in thecollection or engineering chamber or first location in the vessel(thereby also immobilizing the captured target) as the solutions flowthrough or mixes around. As illustrated in FIG. 1(C), the capturedtarget is then engineered either by attaching a plurality of activeagents (e.g., antigenic peptides) to the target surface or loading thetarget with drugs, chemicals, nucleotides, or other bioactive agents(e.g., CRISPR Cas9). For surface modification, the immobilizedbead/target complex is washed and incubated in the vessel with buffersolution containing a plurality of active agents having at least onemoiety that has specificity for a surface protein presented on thesurface of the target. Preferably, the active agents or moieties forsurface loading are of the same “type” of compound (e.g., comprise thesame oligopeptide) selected for the affinity probe used in theimmunomagnetic particles. The immobilized bead/target complex isincubated with the active agents for a period of time sufficient for theactive agents to interact with the captured target. Preferably, theactive agent loading into the vessel is at a concentration such thatsubstantially the entire surface of the target is coated with activeagents (e.g., preferably, substantially all of the target surfaceprotein is bound by active agent).

Instead of surface modification, FIG. 1(C) also depicts an alternativewhere active agents can be loaded into the target. This can be carriedout by washing and incubating the immobilized bead/target complex in themicrofluidic device with buffer solution containing active agents to beloaded, along with detergents or chemical transfection reagents toinduce pore formation in the target for active agent loading, followedby washing with buffer to remove the reagents and close the pores. Otherapproaches for membrane permeabilization can be used, includingirradiation, electroporation, ultrasound, sonication, microinjectionbiolistic particle delivery, and the like, which induce pore formationin the extracellular vesicle membrane other otherwise allow the activeagent to be loaded or introduced into the vesicle. Various active agentscan be loaded including proteins, peptides, nucleic acids (DNA, RNA,oligonucleotides), small molecule drugs, nanoparticles, biologics, andthe like.

In either embodiment, excess active agent is then washed away leavingengineered target immobilized with the immunomagnetic bead.

With reference to FIG. 2A, the engineered target can then be released byexposing the immobilized bead/target complex to activating radiation(e.g., light) of the appropriate wavelength to cleave the photocleavablelinker. This process releases the engineered target along with theaffinity probe which remains bound to the target, which now acts as anengineered carrier or delivery vehicle for the active agents decoratedon the surface of the target. Likewise, in FIG. 2B, the samephotorelease process can be used to release the targets internallyloaded with active agents. A wash buffer can be introduced into thevessel or into the microfluidic device to transport the released targetsdownstream for collection at the outlet of the microfluidic device.Advantageously, the light release step in embodiments of the inventionis carried out with exposure times of 15 minutes or less, preferablyabout 13 minutes or less, more preferably about 12 minutes or less. Asdemonstrated in the working examples, approximately 100% of the capturedtarget is preferably released/cleaved within about 10 minutes ofexposure time.

It will be appreciated that since the magnetic beads are alreadyimmobilized in the engineering chamber or vessel, a separate step is notrequired to separate the target from the magnetic beads in the solution.Rather, upon exposure, the linker between the captured target and thebead is cleaved, thereby releasing the engineered targets, which flowdownstream away from the immobilized beads to the outlet of themicrofluidic device, or can be poured or pipetted away from theimmobilized beads. The released targets, which have been engineered withthe active moieties (aka the “engineered carrier”), can then becollected for analysis and therapeutic use from the outlet of themicrofluidic device or otherwise directly diverted to a further chamberor collection vessel. It will be appreciated that the immunomagneticbeads can then be subsequently collected for re-use by removing themagnetic field from the microfluidic device, such that theimmunomagnetic beads are no longer magnetically immobilized. The beadscan be washed downstream and collected from the outlet.

In some embodiments, the released targets, which have been engineeredwith the attached targeting moieties can be further processed to releasethe targeting moieties from the extracellular vesicle surface. This canbe accomplished without damaging the captured extracellular vesicles,such as by using conventional Western blot stripping and washing buffersto release the affinity probe, and incubating the captured extracellularvesicles with the appropriate stripping buffer, followed by washing toseparate the stripped (bare) extracellular vesicles and theformerly-attached targeting moieties.

As noted, this process can advantageously take place in any suitablevessel, including in a microfluidic device, and as a continuous,integrated, in-line approach for isolating, capturing, engineering, andreleasing intact targets, such as exosomes from a sample, as therapeuticcarriers or delivery vehicles.

Devices

Extracellular vesicles (≤1 μm), particularly exosomes (30-150 nm), arethe emerging cargo for mediating cellular signal transductions. However,standard benchtop methods (e.g., ultracentrifugation and filtration)lack the ability to process immunogenic exosomes specifically amongother microvesicle subtypes, due to time-consuming (>10 h) and extremelytedious isolation protocols. In one aspect, the present disclosureaddresses needs in the art by providing a protocol that can be carriedout using conventional liquid handling vessels and utilizing innovatingmagnetic immobilization and photorelease to streamline the process.

In a further aspect, the present disclosure addresses needs in the artby providing devices that introduce a streamlined microfluidic platformfor harvesting, antigenic modification and photo-release of immunogenicextracellular vesicles and exosomes directly from on-chip culturedcellular media. These devices provide automatic and rapid cell-cultureproduction of antigenic exosomes that can be used in immunotherapy suchas cancer immunotherapy. In one aspect, the devices disclosed hereinenables real-time harvesting and antigenic modification of exosomes withsubsequent photo-release downstream on-demand, as depicted in theoverview in FIG. 3.

Turning now to FIG. 4, disclosed herein is a microfluidic device (200)comprising a cell culture chamber (210) dimensioned to maintainbiological material in a three-dimensional configuration; a mixingchannel (220) fluidly connected to the cell culture chamber andcomprising a plurality of sample inlet channels (222, 224, 226) disposedalong the mixing channel, wherein the ratio of a width of the cellculture chamber (210) to the largest cross-sectional dimension of themixing channel (220) is at least 5:1; an isolation channel (230)defining a path for fluid flow from the mixing channel (220) to anisolation outlet (234); and a collection chamber (240) fluidly connectedto the isolation outlet (234) and comprises a magnet operatively coupledto the collection chamber to produce a magnetic field within thecollection chamber (240).

Devices of the present disclosure can be described by sizes andcomparisons of sizes (e.g., ratios) of components within the device. Insome embodiments, the cell culture chamber (210) has a volume of about200 microliters or greater (for example, 200 microliters or greater, 250microliters or greater, 300 microliters or greater, 350 microliters orgreater, 400 microliters or greater, 450 microliters or greater, 500microliters or greater, 550 microliters or greater, 600 microliters orgreater, 650 microliters or greater, 700 microliters or greater, 750microliters or greater, 800 microliters or greater, 850 microliters orgreater, 900 microliters or greater, 950 microliters or greater, or 1milliliter or greater). In some embodiments, the cell culture chamber(210) has a volume of about 1000 microliters or less (for example, 950microliters or less, 900 microliters or less, 850 microliters or less,800 microliters or less, 750 microliters or less, 700 microliters orless, 650 microliters or less, 600 microliters or less, 650 microlitersor less, 550 microliters or less, 500 microliters or less, 450microliters or less, 400 microliters or less, 350 microliters or less,300 microliters or less, 250 microliters or less, or 200 microliters orless). In some embodiments, the cell culture chamber (210) has a volumeof from about 200 microliters to about 1 milliliter (for example, from200 microliters to 900 microliters, from 200 microliters to 750microliters, from 200 microliters to 500 microliters, from 300microliters to 750 microliters, or from 350 microliters to about 500microliters). In some embodiments, the cell culture chamber has asufficient volume such that the top can be left open for applying a plug(such as a PDMS-made, finger-push plug) for fluid exchange and pushingthe fluid to downstream collection channels.

In some embodiments, the cell culture chamber has a height and a width.The cell culture chamber can have a height of at least 500 microns (forexample, 500 microns or greater, 600 microns or greater, 650 microns orgreater, 700 microns or greater, 750 microns or greater, 800 microns orgreater, 850 microns or greater, 900 microns or greater, 950 microns orgreater, or 1000 microns of greater). In some embodiments, the cellculture chamber has a height of 1000 microns or less (for example, 950microns or less, 900 microns or less, 850 microns or less, 800 micronsor less, 750 microns or less, 700 microns or less, 650 microns or less,600 microns or less, or 500 microns or less). In some embodiments, thecell culture chamber has a height of from 500 microns to 1000 microns(for example, from 600 microns to 1000 microns, from 750 microns to 1000microns, or from 800 microns to 1000 microns).

The cell culture chamber can have a width of at least 200 microns (forexample, 250 microns or greater, 275 microns or greater, 300 microns orgreater, 350 microns or greater, 400 microns or greater, 450 microns orgreater, 500 microns or greater, 550 microns or greater, or 600 micronsor greater). In some embodiments, the cell culture chamber has a widthof 1000 microns or less (for example, less than 1000 microns, 750microns or less, less than 750 microns, 600 microns or less, 550 micronsor less, or 500 microns or less). In some embodiments, the cell culturechamber has a width of from 250 microns to 1000 microns (for example,from 250 microns to 750 microns, from 250 microns to 500 microns, from300 microns to 750 microns, or from 300 microns to 500 microns).

As described herein, the mixing channel (220) fluidly connects to thecell culture chamber and comprises a plurality of sample inlet channels(222, 224, 226) disposed along the mixing channel. The plurality ofsample inlet channels can include a cell culture inlet channel (alsoreferred to herein as B-inlet, 222) that fluidly connects to the cellculture chamber and defines a path for introducing fluid from the cellculture chamber into the mixing channel. The plurality of sample inletchannels can further include a particle inlet channel (also referred toherein as A-inlet, 224) that defines a path for introducing particlesinto the mixing channel. The plurality of sample inlet channels canfurther include a fluid inlet channel (also referred to herein asC-inlet, 226) that defines a path for introducing fluid (such as a washbuffer) into the mixing channel. The plurality of sample inlet channelscan be in any arrangement. For example, the cell culture inlet channelcan be upstream of the particle inlet channel which is upstream of thefluid inlet channel. In other examples, the particle inlet channel canbe upstream of the cell culture inlet channel which is upstream of thefluid inlet channel.

The cell culture inlet channel, the particle inlet channel, and thefluid inlet channel can fluidly converge at a mixing intersection. Thecell culture inlet channel forms a path for fluid flow from the cellculture chamber to the mixing intersection. The particle inlet channelforms a path for fluid flow from a particle inlet to the mixingintersection. The fluid inlet channel forms a path for fluid flow from afluid inlet to the mixing intersection. As used herein, a path of fluidflow can be represented pictorially in the figures by an arrow toindicate the direction of fluid flow through the path of fluid flow.

The mixing channel comprising the cell culture inlet channel, theparticle inlet channel, and the fluid inlet channel has a height and awidth. In some embodiments, the mixing channel has a height of at least50 microns (for example, 75 microns or greater, 100 microns or greater,120 microns or greater, 150 microns or greater, 175 microns or greater,200 microns or greater, 250 microns or greater, 300 microns or greater,350 microns or greater, 400 microns or greater, or 500 microns orgreater). In some embodiments, the mixing channel has a height of 500microns or less (for example, less than 500 microns, 450 microns orless, 400 microns or less, less than 400 microns, 350 microns or less,300 microns or less, less than 300 microns, 275 microns or less, 250microns or less, 200 microns or less, 150 microns or less, 100 micronsor less, or 50 microns or less). In some embodiments, the mixing channelhas a height of from 50 microns to 500 microns (for example, from 100microns to 500 microns, from 200 microns to 500 microns, from 100microns to 350 microns, or from 200 microns to 500 microns).

The mixing channel can have a width of at least 50 microns (for example,75 microns or greater, 100 microns or greater, 120 microns or greater,150 microns or greater, 175 microns or greater, 200 microns or greater,250 microns or greater, 300 microns or greater, 350 microns or greater,400 microns or greater, or 500 microns or greater). In some embodiments,the mixing channel has a width of 500 microns or less (for example, lessthan 500 microns, 450 microns or less, 400 microns or less, less than400 microns, 350 microns or less, 300 microns or less, less than 300microns, 275 microns or less, 250 microns or less, 200 microns or less,150 microns or less, 100 microns or less, or 50 microns or less). Insome embodiments, the mixing channel has a width of from 50 microns to500 microns (for example, from 100 microns to 500 microns, from 200microns to 500 microns, from 100 microns to 350 microns, or from 200microns to 500 microns).

The ratio of the culture chamber width to the to the largestcross-sectional dimension of the mixing channel is at least 2:1, atleast 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, atleast 8:1, at least 9:1, at least 10:1, at least 12:1, at least 15:1, atleast 18:1, at least 20:1, at least 25:1, at least 50:1, at least 75:1,at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least300:1, at least 350:1, at least 400:1, at least 450:1, or at least500:1. In some embodiments, the ratio of the culture chamber width tothe largest cross-sectional dimension of the mixing channel is from 5:1to 500:1, from 5:1 to 200:1, from 5:1 to 100:1, from 2:1 to 25:1, from5:1 to 20:1, from 5:1 to 15:1, from 6:1 to 25:1, from 6:1 to 20:1, from6:1 to 12:1, from 8:1 to 25:1, or from 10:1 to 25:1. A ratio of theculture chamber width to the largest cross-sectional dimension of themixing channel which is greater than one (e.g., 2:1) defines a narrowingof channel width at the mixing channel inlet.

One or more channels in the microfluidic device can, in someembodiments, comprise a fluid mixing mechanism which facilitates themixing of fluids flowing through the device. A fluid mixing mechanisminduces turbulent flow so as to mix flowing fluids. Suitable mixingmechanisms include a serpentine or tortuous channel, a channelprotrusion or indentation, a channel curvature, among other knownmechanisms.

In some embodiments, a fluid mixing mechanism can be present in themixing channel and/or in an isolation channel of the microfluidicdevice. For example, the microfluidic device can include an isolationchannel that fluidly connects to the mixing channel to an isolationoutlet. The isolation channel can form a part of the mixing channel orcan be separate. In some embodiments, the isolation channel defines apath for fluid flow from the mixing channel to an isolation outlet. Theisolation channel can comprise a serpentine geometry which enhancesmixing as the fluids combine. Referring to FIG. 4, an isolation channel(230) having a serpentine geometry can be positioned within the deviceat a location advantageous for fluid mixing, for example, between amixing intersection and a collection chamber. In some embodiments, theisolation channel is positioned immediately adjacent a mixingintersection (e.g., immediately downstream).

The isolation channel can have a similar or narrowed width as comparedto the mixing channel width. In some embodiments, the isolation channelcan have a narrowed width as compared to the mixing channel width by atleast several means. For example, the isolation channel can comprise oneor more channel constriction domains (see, for example, the channelconstriction domains (232) which form the narrowed width in theisolation channel in FIG. 4) disposed within the isolation channelbetween the isolation channel inlet and the isolation channel outlet.The channel constriction domains can produce a local vortex flow profileof fluid flowing in the device. In some embodiments, the isolationchannel comprises at least one, at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten channel constriction domains. Any one ormore channel constriction domains can be a protrusion and/or indentationin the channel sidewalls. The protrusion and/or indentation can have anyshape, for example rounded, linear, triangular, irregular, etc. Theprotrusion and/or indentation can encircle the inner walls of thechannel (e.g., as a ring), or one or more protrusions and/or indentationcan be positioned on one or more inner sidewalls of the channel.Inclusion of a channel constriction domain can increase fluid flowturbulence and fluid mixing, where desirable.

Devices disclosed herein can include a collection chamber (240) fluidlyconnected to the isolation outlet (234). In some embodiments, the devicecan further comprise a magnet operatively coupled to the collectionchamber to produce a magnetic field within the collection chamber. Themagnet can be any magnet capable of providing a magnetic field withinthe collection chamber. In some embodiments, the magnetic field caninclude an oscillating magnetic field. An oscillating magnetic field isa magnetic field which varies regularly (e.g., automated periodicregularity) or irregularly (e.g., by user-based controls) over time. Anoscillating magnetic field includes dynamic changes in the spatialorientation of the north and south magnetic poles, such that thedirection of the magnetic field changes over time. Such changes can becyclical or irregular. Inclusion of an oscillating magnet capable ofproviding an oscillating magnetic field within the collection chambercan induce magnetic probes (e.g., magnetic beads or particles) withinthe collection chamber to dynamically move inside the collection chamberalong a direction of the magnetic field. As the direction of themagnetic field changes, the directional movement of magnetic particleswithin the collection chamber also changes. This can be used to fosterinteraction (and association/binding) between the magnetic particles andtargets present in a fluid in the collection chamber. In someembodiments, the magnetic field can be obtained from a permanent magnet.The permanent magnet can be removed when not in use, for example, toswitch off the magnetic field.

In some embodiments, the magnet can have any shape such as a toroidalshape. In some embodiments, the magnet comprises a Helmholtz coil or apermanent magnet.

The magnetic field can be present over the entire width of thecollection chamber. In some embodiments, the magnetic field can be overa portion of the collection chamber, for example over at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95% ofthe entire width of the collection chamber. Further, the magnetic fieldcan be over other portions of the microfluidic device. For example, themagnetic field can be over one or more of a portion of or the entiretyof the mixing channel, the mixing intersection, the particle inletchannel, or other components.

In some embodiments, the device can comprise a pump operably coupled tothe microfluidic device. The pump can be any pump known in the artcapable of inducing fluid flow within the device. In some embodiments,the pump can impart negative pressure within the device, thereby pullingfluid through a channel. Examples of suitable pumps can be found inUS20170065978 and US20170001197, each of which are incorporated byreference in their entireties.

Turning now to FIG. 6, also disclosed is a microfluidic device (2000)comprising a cell culture chamber (2100) comprising a cell culture inlet(2102) and a cell culture outlet (2104), a fluid inlet channel (2202)and a particle inlet channel (2204), wherein the cell culture outlet(2104), the fluid inlet channel (2202), and the particle inlet channel(2204) fluidly converge at a mixing intersection (2200); a mixingchannel (2300) fluidly connected to the mixing intersection (2200) anddefining a path for fluid flow from the mixing intersection to a mixingoutlet, wherein the ratio of a width of the cell culture chamber (2100)to the largest cross-sectional dimension of the mixing channel (2300) isat least 5:1; and a collection chamber (2400) fluidly connected to themixing outlet and comprises a magnet operatively coupled to thecollection chamber (2400) to produce a magnetic field within thecollection chamber. The mixing channel (2300) can comprise an isolationchannel (2302) disposed between the mixing intersection (2200) and themixing outlet.

Another aspect of the microfluidic devices provided herein relates tomultiplexed microfluidic devices which contain two or more sets ofchambers and/or channels including the cell culture chamber, the mixingchannel, the isolation channel, and the collection chamber. Configuringtwo or more of such channels and/or chambers on a single microfluidicdevice can increase sample-processing throughput and/or allow forparallel processing of at least two samples or portions of the samplefor different fractions or manipulations. Two or more chambers and/orchannels can be arranged in series, in parallel or in a combinationthereof.

In some embodiments of a parallel multiplexed microfluidic device, twoor more mixing channels can have separated sample inlets disposed on thesame microfluidic device. Such arrangement can be employed for multiplefluid samples. Alternatively, the plurality of the mixing channels canbe connected to the same sample inlets for parallel processing of thesame fluid sample. Additionally, the two or more mixing channels canhave separated outlets disposed on the same microfluidic device or beconnected to the same outlet. In one or more embodiments, multiplexedmicrofluidic devices are contemplated having as many as 96 sampleinlets.

The microfluidic devices of the present disclosure can be used incombination with the various compositions, devices, methods, products,and applications disclosed herein. In some embodiments, the microfluidicdevices can be a stand-alone microfluidic device. In some embodiments,one or more microfluidic devices can be integrated as part of anequipment, a module or a system. In other embodiments, one or moremicrofluidic devices can be fluidically coupled to an equipment, amodule or a system.

By way of example only, one or more microfluidic device and/ormultiplexed microfluidic devices can be fluidically coupled to adetection module. As used herein, the term “fluidically coupled” refersto two or more devices and/or modules connected in an appropriate mannersuch that a fluid can pass or flow from one device or module to theother device or module. When two or more devices and/or modules arefluidically coupled together, additional devices and/or modules can bepresent between the two or more devices and/or modules.

Alternatively, two the two or more devices and/or modules can beconnected such that a fluid can pass or flow directly from a firstdevice or module to a second device or module without any interveningdevices or modules. Two or more devices or modules can be fluidicallycoupled, for example, by connecting an outlet of a first device ormodule to an inlet of a second device or module using tubing, a conduit,a channel, piping or any combinations thereof.

The detection module can perform any method of detection disclosedherein or other methods known in the art. In some embodiments, thedetection module can include a sample-treatment module before the sampleis detected for analysis. For example, the exosomes (including orexcluding the immunomagnetic particles) can be subjected toimmunostaining before detection by microscopy. Examples of the detectionmodule can include, without limitations, a microscope (e.g., abrightfield microscope, a fluorescence microscope, or a confocalmicroscope), a spectrophotometer (e.g., UV-Vis spectrophotometer), acell counter, a biocavity laser (see, e.g., Gourley et al., J. Phys. D:Appl. Phys. 36: R228-R239 (2003)), a mass spectrometer, an imagingsystem, an affinity column, a particle sorter, e.g., a fluorescentactivated cell sorter, capillary electrophoresis, a sample storagedevice, and sample preparation device. In some embodiments, a computersystem can be connected to the detection module, e.g., to facilitate theprocess of sample treatment, detection and/or analysis.

Moreover, although depicted with respect to particular microfluidichandling devices, it will be appreciated that the protocol can belikewise be implemented in various other liquid handling vessels asexemplified in the working examples.

Methods of Making

The devices described herein can be made of any material that iscompatible with a fluid sample. In some embodiments, the material forfabrication of the devices described herein can be penetrated by amagnetic field. In some embodiments, the material for fabrication of thedevices described herein can be substantially transparent so that thesample therein can be photocleaved in situ or it can be viewed under amicroscope, e.g., for in situ analysis of the magnetically-labeledexosomes. Exemplary materials that can be used to fabricate themicrofluidic devices described herein can include, but are not limitedto, glass, co-polymer, polymer or any combinations thereof. Exemplarypolymers include, but are not limited to, polyurethanes, rubber, moldedplastic, polymethylmethacrylate (PMMA), polycarbonate,polytetrafluoroethylene (TEFLON™) polyvinylchloride (PVC),polydimethylsiloxane (PDMS), polysulfone, and ether-based, aliphaticpolyurethane.

The methods used in fabrication of any embodiments of the microfluidicdevices described herein can vary with the materials used, and include3D printing methods, soft lithography methods, microassembly, bulkmicromachining methods, surface micro-machining methods, standardlithographic methods, wet etching, reactive ion etching, plasma etching,stereolithography and laser chemical three-dimensional writing methods,solid-object printing, machining, modular assembly methods, replicamolding methods, injection molding methods, hot molding methods, laserablation methods, combinations of methods, and other methods known inthe art.

In specific embodiments, the microfluidic devices described herein canbe fabricated using a 3D printer. For example, methods of making themicrofluidic devices can include providing three pieces of PDMS moldsincluding a base, wall, and top magnet holder as shown in FIG. 7. Themold can be printed out by a 3D printer. The molds can be coated withSportline palladium at a thickness of 20 nm followed by assembly usingmethods known in the art. The PDMS cell chamber can be sized so thatwhen it is filled, the cell culture chamber has an open end for chamberplug. PDMS can be cast by a 10:1 ratio with a linker reagent andincubated at a temperature of 40° C. for 6 hours. After the PDMS iscured, it can be peeled out easily. Chip inlets and outlet can bepunched by using puncher. The PDMS chips can then be post-bond on a hotpad at the temperature of 40° C. for 5 mins. The chips can be cleanedusing DI water, and sterilized by autoclave (at 121° C. for 30 mins).

Methods of Use

As discussed herein, the process and associated devices can be used forisolating, capturing, engineering, and releasing engineeredextracellular vesicles and various vesicle-like biological structures.In certain embodiments, the engineered extracellular vesicles areimmunogenic exosomes. As used herein, the term “exosome” generallyrefers to externally released vesicles originating from the endosomiccompartment or any cells, e.g., tumor cells (e.g., prostate cancercells), and immune cells (e.g., antigen presenting cells, such asdendritic cells, macrophages, mast cells, T lymphocytes or Blymphocytes). Exosomes are generally membrane vesicles with a size ofabout 20-150 nm that are released from a variety of different cell typesincluding tumor cells, red blood cells, platelets, lymphocytes, anddendritic cells. Exosomes can be formed by invagination and budding fromthe membrane of late endosomes. They can accumulate in cytosolicmultivesicular bodies (MVBs) from where they can be released by fusionwith the plasma membrane. Without wishing to be bound by theory, theprocess of vesicle shedding is particularly active in proliferatingcells, such as cancer cells, where the release can occur continuously.When released from tumor cells, exosomes can promote invasion andmigration. Thus, in some embodiments, the immunomagnetic particlesdescribed herein can be used to capture exosomes originating from cancercells. Depending on the cellular origin, exosomes can recruit variouscellular proteins that can be different from the plasma membraneincluding MHC molecules, tetraspanins, adhesion molecules andmetalloproteinases. Among many subtypes of exosomes, the immunogenicexosomes with an intrinsic payload of MHC class I and II molecules andother co-stimulatory molecules are able to mediate immune responses,which opens up opportunities for the development of novel cancervaccines and delivery in immunotherapy.

Accordingly, also provided herein are methods of producing immunogenicexosomes in a microfluidic device or other suitable vessel disclosedherein. The methods of producing immunogenic exosome complexes cancomprise introducing cells into a suitable vessel, or into the cellculture chamber of the microfluidic device. The cells can include anycells from which extracellular vesicles can be obtained. Such cellsinclude dendritic cells, stem cells, immune cells, megakaryocyteprogenitor cells, macrophages, or combinations thereof.

The method for producing immunogenic exosomes can further includeculturing the cells under conditions allowing release of exosomes. Insome embodiments, the methods can include enriching or expanding thenumber of exosomes present in the cell sample through mediating theirparent cell growth using stimulations known in the art. Conventionalmethods for culturing a parent cell to produce exosomes are known in theart and can be used in the methods disclosed herein. In someembodiments, the cells can be cultured for a period of time, e.g., atleast about 30 mins, at least about 45 mins, at least about 1 hour, atleast about 2 hours, at least about 3 hour, at least about 5 hours, atleast about 6 hours, at least about 8 hours, at least about 10 hours, atleast about 12 hours, at least about 15 hours, at least about 18 hours,at least about 20 hours, at least about 24 hours, at least about 30hours, at least about 36 hours, at least about 40 hours, or at leastabout 48 hours.

The method for producing immunogenic exosomes complex can furthercomprise mixing the cell culture comprising exosomes with immunomagneticparticles for capturing the exosomes and a wash solution to form amixture. In some embodiments, the methods include introducing theexosomes from the cell culture into a mixing channel and mixing theexosomes with immunomagnetic particles and a wash buffer to form amixture. The immunomagnetic particles can be introduced into the mixingchannel via the particle inlet channel and the wash buffer can beintroduced into the mixing channel via the fluid inlet channel.

The immunomagnetic particles can selectively bind to the exosomespresent in the cell culture to form exosome-bound immunomagneticparticles. Accordingly, the method can include allowing the exosomes toreact with the immunomagnetic particles. The immunomagnetic particlescan include a magnetic particle and be of any shape, including but notlimited to spherical, rod, elliptical, cylindrical, and disc. In someembodiments, magnetic particles having a substantially spherical shapeand defined surface chemistry can be used to minimize chemicalagglutination and non-specific binding. As used herein, the term“magnetic particles” can refer to a nano- or micro-scale particle thatis attracted or repelled by a magnetic field gradient or has a non-zeromagnetic susceptibility. The magnetic particles can be ferromagnetic,paramagnetic or super-paramagnetic. In some embodiments, magneticparticles can be super-paramagnetic.

The magnetic particles can range in size from 1 nm to 5 microns. Forexample, magnetic particles can be about 500 nm to about 5 microns insize. In some embodiments, magnetic particles can be about 1 micron toabout 5 microns in size. In some embodiments, magnetic particles can beabout 1 micron to about 3 microns in size. Magnetic particles are aclass of particles which can be manipulated using magnetic field and/ormagnetic field gradient. Such particles commonly consist of magneticelements such as iron, nickel and cobalt and their oxide compounds.Magnetic particles (including nanoparticles or microparticles) arewell-known and methods for their preparation have been described in theart. Magnetic particles are also widely and commercially available. Aparticularly preferred particle is a magnetic particle having agraphene-oxide layer or coating which is comprised of graphene-oxidenanosheets, as described in US 2018/0100853, filed Oct. 9, 2017,incorporated by reference in its entirety herein.

The magnetic particles can be coated with a plurality of linkerscomprising respective affinity probes (molecules) for capturing thetarget (such as an antigen peptide or antigenic epitope thereof) havingno adverse effect on the magnetic property. In this regard, the magneticparticle can be functionalized with an organic moiety or functionalgroup and photocleavable linker that can connect the magnetic particleto respective affinity probes for capturing the exosomes. Such organicmoiety or functional groups can typically comprise a direct bond or anatom such as oxygen or sulfur, a unit such as amino groups, carboxylicacid groups, epoxy groups, tosyl groups, silica-like groups, carbonylgroups, amide groups, SO, SO₂, SO₂NH, SS, or a chain of atoms.

In certain embodiments, the magnetic particles can be coated with onemember of an affinity binding pair that can facilitate the conjugationof the magnetic particles to the affinity probe for capturing theexosomes. The term “affinity binding pair” or “binding pair” refers tofirst and second molecules that specifically bind to each other. Onemember of the binding pair is conjugated with first part to be linkedwhile the second member is conjugated with the second part to be linked.Exemplary binding pairs include any haptenic or antigenic compound incombination with a corresponding antibody or binding portion or fragmentthereof (e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulinand goat antimouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin, hormone[e.g., thyroxine and cortisol-hormone binding protein, receptor-receptoragonist, receptor-receptor antagonist (e.g., acetylcholinereceptor-acetylcholine or an analog thereof), IgG-protein A, IgG-proteinG, IgG-synthesized protein AG, lectin-carbohydrate, enzyme-enzymecofactor, enzyme-enzyme inhibitor, and complementary oligonucleotidepairs capable of forming nucleic acid duplexes), and the like. Thebinding pair can also include a first molecule which is negativelycharged and a second molecule which is positively charged.

One example of using binding pair conjugation is the biotin-avidin,biotin-streptavidin or biotin-neutravidin conjugation. Accordingly, insome embodiments, the magnetic particles can be coated with avidin-likemolecules (e.g., streptavidin or neutravidin), which can be conjugatedto biotinylated linkages for use as capturing molecules.

In some embodiments, the magnetic particles can be furtherfunctionalized with a cleavable chemical moiety that can link themagnetic particles to the affinity probe for capturing the exosomes, andis susceptible to an externally-applied cleavage agent/conditions, e.g.,UV light, pH, redox potential or the presence of degradative moleculessuch as enzymes. In specific examples, the cleavable linker can beconjugated to a member of a binding pair (such as biotin) at onefunctional end to link to the magnetic particles, and the otherfunctional end provides an affinity probe for capturing exosomes. Thus,after the exosomes bound magnetic particles are separated from a fluidsample, the exosomes can be separated from the magnetic particles, ifneeded, by cleaving the cleavable chemical moiety between the magneticparticles and the affinity probe.

Exemplary cleavable linking groups include, but are not limited to,photocleavable and redox cleavable linking groups (e.g., —OC(O)NH—,—S—S—, and —C(R)₂—S—S—, wherein R is H or C₁-C₆ alkyl); phosphate-basedcleavable linking groups (e.g., —O—P(O)(OR)—O—, —O—P(S)(OR)—O—, and—O—P(S)(H)—S—, wherein R is optionally substituted linear or branchedC₁-C₁₀ alkyl); acid cleavable linking groups (e.g., hydrazones, esters,and esters of amino acids, —C═NN— and —OC(O)—); ester-based cleavablelinking groups (e.g., —C(O)O—); peptide-based cleavable linking groups,(e.g., linking groups that are cleaved by enzymes such as peptidases andproteases in cells, e.g., —NHCHR_(A)C(O)NHCHR_(B)C(O)—, where R_(A) andR_(B) are the R groups of the two adjacent amino acids).

In some embodiments, the cleavable linking group is a photocleavablegroup that can be cleaved by UV light. Specific examples ofphotocleavable groups include ortho nitrobenzyl derivatives andbenzylsulfonyl such as 6-nitroveratryloxycarbonyl (NVOC),2-nitrobenzyloxycarbonyl (NBOC), α,α-dimethyl-dimethoxybenzyloxycarbonyl(DDZ), ortho-nitrobenzyl (ONB), 1-(2-nitrophenyl)ethyl (NPE),alpha-carboxy-2-nitrobenzyl (CNB), 4,5-dimethoxy-2-nitrobenzyl (DMNB),1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE),5-carboxymethoxy-2-nitrobenzyl (CMNB), and(5-carboxymethoxy-2-nitrobenzyl)oxy)carbonyl (CMNCBZ). It will beappreciated that the substituents on the aromatic core are selected totailor the wavelength of absorption, with electron donating groups(e.g., methoxy) generally leading to longer wavelength absorption. Forexample, nitrobenzyl (NB) and nitrophenylethyl (NPE) are modified byaddition of two methoxy residues into 4,5-dimethoxy-2-nitrobenzyl and1-(4,5-dimethoxy-2-nitrophenyl)ethyl, respectively, thereby increasingthe absorption wavelength range to 340-360 nm. Additional examples ofthe photoremovable protecting groups include multiply substituted nitroaromatic compounds containing a benzylic hydrogen ortho to the nitrogroup, wherein the substituent may include alkoxy, alkyl, halo, aryl,alkenyl, nitro, halo, or hydrogen. Other materials which may be usedinclude o-hydroxy-α-methyl cinnamoyl derivatives, photocleavable groupsbased on the coumarin system, such as BHC (such as described in U.S.Pat. No. 6,472,541, the disclosure of which is incorporated by referenceherein), photocleavable group comprising the pHP group (such asdescribed in Givens et al., J. Am. Chem. Soc. 122 2687-2697 (2000), thedisclosure of which is incorporated by reference herein), ketoprofenderived linkers, other ortho-nitro aromatic core scaffolds include thosethat trap nitroso byproducts in a hetero Diels Alder reaction (generallydiscussed in U.S. Patent Application No. 2010/0105120, the disclosure ofwhich is incorporated by reference herein), nitrodibenzofurane (NDBF)chromophore, or a diazo-azide. Further examples of photocleavable groupsmay be found in, for example, Patchornik, J. Am. Chem. Soc. (1970)92:6333 and Amit et al., J. Org. Chem. (1974) 39:192, the disclosures ofwhich are incorporated by reference herein.

As discussed above, a photocleavable group is one whose covalentattachment to a molecule (such as to a member of a binding pair examplebiotin at one functional end and the other functional end to an affinityprobe) is cleaved by exposure to light of an appropriate wavelength. Inone aspect, release of the affinity probe and/or binding pair occurswhen the conjugate is subjected to ultraviolet light or near ultravioletlight. For example, photorelease of the affinity probe may occur at awavelength ranging from about 200 to 380 nm (the exact wavelength orwavelength range will depend on the specific photocleavable group used,and could be, for example, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, or 380 or some rangetherebetween). In another aspect, release of the affinity probe mayoccur when the conjugate is subjected to visible light. For example,photorelease of the affinity probe may occur at a wavelength rangingfrom about 380 to 780 nm (the exact wavelength or wavelength range willdepend on the specific photocleavable group used, and could be, forexample, 380, 400, 450, 500, 550, 600, 650, 700, 750, or 780, or somerange therebetween).

As described herein, the magnetic particles further comprise an affinityprobe (also referred to herein as a molecule for capturing the exosomesor capturing molecule). As used herein, the term “affinity probe” or“capturing molecule” refers to any molecule, cell or particulatematerial. Suitable affinity probes comprising a magnetic particle aredescribed in US20170065978 and US20170001197, each of which areincorporated by reference in their entireties. The affinity probes cancomprise a binding element which specifically binds the target (exosomeor other extracellular vesicles) of interest. For example, the bindingelement can be a nucleic acid oligomer, antibody, enzyme, hormone,growth factor, cytokine (e.g., inflammatory cytokines), proteins,peptide, prion, lectin, oligonucleotide, carbohydrate, lipid, molecularand chemical toxin or other binding element which has high affinity andhigh specificity for the target, and specificity for a designatedsurface protein on the target. One or more binding elements (e.g., apeptide) can be attached to the magnetic particle via the cleavablelinker by methods known in the art. Generally, a binding element has anaffinity constant (Ka) greater than about 10⁵ M⁻¹ (e.g., 10⁶ M⁻¹, 10⁷M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, and 10¹² M⁻¹ or more) withthe target, particularly exosome or other extracellular vesicles.

In certain embodiments, the affinity probe includes an antigenic peptideor antigenic epitope thereof. As used herein, the term “antigens” refersto a molecule or a portion of a molecule capable of being bound by aselective binding agent, such as an antibody, and additionally capableof being used in an animal to elicit the production of antibodiescapable of binding to an epitope of that antigen. An antigen may haveone or more epitopes. The term “antigen” can also refer to a moleculecapable of being bound by an antibody or a T cell receptor (TCR) ifpresented by MHC molecules. The term “antigen,” as used herein, alsoencompasses T-cell epitopes. An antigen is additionally capable of beingrecognized by the immune system and/or being capable of inducing ahumoral immune response and/or cellular immune response leading to theactivation of B- and/or T-lymphocytes. An antigen can have one or moreepitopes (B- and T-epitopes). The specific reaction referred to above ismeant to indicate that the antigen will preferably react, typically in ahighly selective manner, with its corresponding antibody or TCR and notwith the multitude of other antibodies or TCRs which may be evoked byother antigens. Antigens as used herein may also be mixtures of severalindividual antigens.

As described above, the affinity probe (antigen) can be a protein or apeptide. In some embodiments, the protein or peptide can be essentiallyany protein that can activate immune cells and/or primeimmune-responses, bind to a rare cell, e.g., a circulating tumor cell, astem cell and/or a microbe. By way of example only, if the targetspecies is cancer, exemplary proteins or peptides or other molecule thatcan be used to generate cancer-affinity probes can include, but are notlimited to, MAGE-A3, gp-100, HER-2, p53, PSA-1, or MART-1, EGFR, ERCC1,CXCR4, EpCAM, CEA, ErbB-2, E-cadherin, mucin-1, cytokeratin, PSA, PSMA,RRM1, androgen receptor, estrogen receptor, progesterone receptor, IGF1,cMET, EML4, or leukocyte associated receptor (LAR).

In some embodiments, the affinity probe can be an antibody or a portionthereof, or an antibody-like molecule. In some embodiments, thecapturing molecule can be an antibody or a portion thereof, or anantibody-like molecule that is specific for detection of a rare-cell,e.g., a circulating tumor cell, a stem cell and/or a microbe biomarker.In some embodiments, the affinity probe can be an aptamer. In someembodiments, the affinity probe can be a DNA or RNA aptamer. In someembodiments, the affinity probe can be a cell surface receptor ligand.Exemplary, cell surface receptor ligand includes, for example, a cellsurface receptor binding peptide, a cell surface receptor bindingglycopeptide, a cell surface receptor binding protein, a cell surfacereceptor binding glycoprotein, a cell surface receptor binding organiccompound, and a cell surface receptor binding drug. Additional cellsurface receptor ligands include, but are not limited to, cytokines,growth factors, hormones, antibodies, and angiogenic factors. In someembodiments, any art-recognized cell surface receptor ligand that canbind to a rare cell, e.g., a circulating tumor cell, a stem cell and/ora microbe, can be used as an affinity probe on the magnetic particlesdescribed herein. In one or more embodiments, an affinity probe isselected to target an immunostimulatory molecule presented on thesurface of the target (e.g., exosome), such as MHC class I molecule, anMHC class II molecule, an interleukin, TNFα, IFNγ, RANTES, G-CSF, M-CSF,IFNα, CTAPIII, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1α,MIP-1β and combinations thereof. More preferably, upon binding of theaffinity probe (and subsequent release of the target), the resultingengineered target comprising the bound affinity probe enhances theimmunogenic potential of the released target. For example, the bindingof an MHC class I surface protein by the affinity probe creates acomplex that will (in therapeutic use) enhance recognition and uptake ofthe engineered target by an antigen presenting cell for stimulation andactivation of the immune system. The peptides are preferably 15 aminoacid residues or less in length, more preferably 13 residues or less inlength, even more preferably 12 residues or even 11 residues or less inlength. Example affinity probes include those listed in the Table below:

SEQ SEQ ID ID NO: NO: Peptide 1: SIINFEKL  1Peptide 2: RSV M2 82-90 peptide  2 sequence SYIGSINNIPeptide 3: Fusion 85-93 peptide  3 Peptide 4: M187-195 peptide  4sequence KYKNAVTEL sequence NAITNAKII MAGE-A1 161-169: EADPTGHSY  5MAGE-A3 168-176: EVDPIGHLY  6 MAGEA-10 254-262:  7MAGEA3 112-120: KVAELVHFL  8 GLYDGMEHL MAGEA1 278-286: KVLEYVIKV  9MAGEA3 271-279: FLWGPRALV 10 MAGEA3 112-120 (alternative 11MAGEA2 157-166: YLQLVFGIEV 12 version): KVAEELVHFL MAGE-A4 230-239: 13MAGE-C1 1083-1091: 14 GVYDGREHTV KVVEFLAML MAGE-C2 191-200: LLFGLALIEV15 MAGE-C2 336-344: ALKDVEERV 16 MAGEA3 97-105: TFPDLESEF 17MAGEA5 5-12: HNTQYCNL 18 Prostate Specific Antigen 146-154: 19Carcinogenic Embryonic Antigen 20 KLQCVDLHV (CEA) 694-702: GVTYACFVSNLCarcinogenic Embryonic Antigen 21 G250 (renal cell carcinoma) 217-225:22 (CEA) 652-660: TYACFVSNL HLSTAFARV HER-2/neu 435-443: ILHNGAYSL 23HER-2/neu 63-71: TYLPTNASL 24 HER-2 434-443: ILHDGAYSL 25Neu/Her-2/Erbb2 proto-oncoprotein 26 66-74: TYVPANASLgp100 (pmel17) 209-217: 27 gp100-intron 4(170-178): 28 IMDQVPFSVVYFFLPDHL gp100 (pmel17) 154-162: 29 gp100 (pmel17) 476-485: 30KTWGQYWQV VLYRYGSFSV gp100 (pmel) 209-217: ITDQVPFSV 31gp100 (pmel) 280-288 (288V): 32 YLEPGPVTV gp100: YLEPGPVTA 33gp100 (pmel17) 25-33: 34 KVPRNQDWL gp100 (pmel17) 17-25: 35gp100-intron 4 (170-178): 36 ALLAVGATK VYFFLPDHLHER-2/neu 369-377: KIFGSLAFL 37 p53 264-272: LLGRNSFEV 38p53 187-197: GLAPPQHLIRV 39 p53 149-157: SLPPPGTRV 40p53 139-147: KLCPVQLWV 41 p53 65-73: RMPEAAPPV 42 p53 103-111: YLGSYGFRL43 Prostatic Acid Phosphatase-3 (PAP- 44 3): FLGYLILGVPSM P2 (prostate): ALFDIESKV 45 Prostate Stem Cell Antigen (PSCA) 4614-22: ALQPGTALL MelanA/MART 26-35: 47Prostate Specific Antigen-1 (PSA-1) 48 ELAGIGILTV 141-150: FLTPKKLQCVMUC-1 12-20: LLLLTVLTV 49 Human Mena protein (overexpressed 50in breast cancer): GLMEEMSAL HER-2/neu 689-697: RLLQETELV 51HER-2/neu (85-94): LIAHNQVRQV 52 Prostate Specific Antigen-1 (PSA-1) 53Prostate Specific Antigen-1 153-161: 54 154-163: VISNDVCAQV CYASGWGSIPSA 65-73: HCIRNKSVI 55 EGF-R-479 350-359: 56 KLFGTSGQKTEGF-R 1138-1147: YLNTVQPTCV 57 VEGFR2 400-408: VILTNPISM 58VEGFR2/KDR fragment 1 614-624: 59 FSNSTNDILI

It will be appreciated that the foregoing peptides, suitable foraffinity probes, are also exemplary of active agents or moieties forsurface loading onto the engineered target.

The immunomagnetic particles (that is magnetic particles bound to theaffinity probe) are preferably formed before the mixing process. Forexample, the affinity probe (e.g., an antigenic peptide bound to biotinvia a cleavable linker) and streptavidin coated magnetic particles aremixed together for an effective period of time for the biotinylatedaffinity probe linkages to substantially completely coat the entiresurface of the avidin coated particles.

Thus, the affinity probe is preferably added to the streptavidin coatedmagnetic particles for a period of time, before adding the exosomecontaining cell culture. In such embodiments, the affinity probe can befirst added to the streptavidin coated magnetic particles for a periodof time sufficient for at least a portion of the added amount ofaffinity probe to bind with the streptavidin coated magnetic particles(and preferably for complete binding of affinity probe so as to coat theentire surface of the particle with probe linkages extending therefrom).

The exosomes present in the exosome containing cell culture are thenadded into the same fluid sample, where the exosomes can bind to theaffinity probe, which have already formed a conjugate with thestreptavidin coated magnetic particles.

In some embodiments, the immunomagnetic particles can be separatelyformed before being introduced into the mixing channel of the device.

The amount of the immunomagnetic particles required to be added into thesample can depend on a number of factors, including, but are not limitedto, volume of the sample to be processed, valency of the magneticparticles available for conjugation with the affinity probe, expectedabundance of the exosomes present, and any combinations thereof. Toohigh amounts of the immunomagnetic particles added into the device caninduce non-specific binding and/or clogging inside the microfluidicdevice. Too low amounts of the immunomagnetic particles can result in alow capture efficiency. One skilled in the art can determine theconcentration of the immunomagnetic particles and capturing molecules.

The exosomes can be allowed to mix with the immunomagnetic particles forany period of time, e.g., seconds, minutes or hours. In someembodiments, the exosomes can be mixed with the immunomagnetic particlesfor at least about 1 min, at least about 2 mins, at least about 5 mins,at least about 10 mins, at least about 15 mins, at least about 30 mins,at least about 1 hour, at least about 2 hours or more. A person havingordinary skill in the art can readily determine an optimum time formixing time, based on a number of factors, including, but not limitedto, the affinity of the immunomagnetic particles with the exosomes,concentrations, mixing temperature and/or mixing speed. However, in oneor more embodiments, exosomes are mixed with the particles for 1 hour orless.

The exosomes and immunomagnetic particles can be introduced into thesample inlets of the microfluidic device at any flow rate that providesa sufficient residence time for the mixture to retain in the mixingchannel and isolation channel of the microfluidic device describedherein. In some embodiments, the samples can be introduced at a flowrate of between 0.1 uL/min to 1 uL/min. The sample fluids can beintroduced into the inlet of the microfluidic device by any methodsknown to a skilled artisan. For example, a flow generator can beconnected to at least one of the inlets and the outlet of themicrofluidic device described herein. Non-limiting examples of a flowgenerator can include a peristaltic pump, a syringe pump and anyart-recognized pump that can be generally used to flow a fluid throughthe microfluidic device.

The method of producing immunogenic engineered targets can furtherinclude capturing the exosomes bound to the immunomagnetic particles byapplying a magnetic field within a collection or engineering chamber. Insome embodiments, the magnet has a strong magnetic field strengthsufficient to create a magnetic field gradient to cause themagnetically-labeled exosomes to separate from the fluid sample in thecollection chamber. The immobilized magnetically-labeled exosomes can beremoved from the microfluidic device or reaction vessel for furtherprocessing. Preferably, the captured exosomes are further engineered andloaded with additional active moieties on the surface or internally asdiscussed herein. Subsequently, the method includes photolyticallycleaving the exosomes bound to the immunomagnetic particles forreleasing intact exosomes coated with active moieties or loadedinternally with active agents.

The released target (exosomes) can be provided as a pharmaceuticalcomposition. The pharmaceutical composition can include the immunogenicexosomes and a pharmaceutically acceptable excipient. It will beappreciated that the active moieties can be tailored to provide either aspecific adaptive immune response against a target condition, or can beselected more generally to activate the innate immune system against avariety of infections or conditions.

The methods described herein can be used to process samples in realtime. For example, the methods allow real-time, continuous harvestingand antigenic modification of exosomes with subsequent photo-releasedownstream on-demand.

As described herein the methods can be used to produce an immunogenicexosome complex or other immunogenic vesicle-like structures. In certainembodiments, the immunogenic exosome complex can comprise an antigenpeptide conjugated to a surface of an exosome. The methods describedherein for making the immunogenic exosome complex provides complexeswith a significantly higher activation rate for T-cells thannon-engineered exosomes. In some examples, the immunogenic exosomecomplex can activate T-cell by at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, or at least 90% compared to a native exosome. Because of theimproved activation rate, the immunogenic complexes described herein canbe used in cancer immunotherapy.

Accordingly, methods of treating disease in a subject using theimmunogenic complex are disclosed. The method can include administeringto the subject a composition comprising an immunogenic complex. In someembodiments, the disease can be an infection. In some examples, thedisease can be cancer. The method can further comprise administering achemotherapeutic agent that has been loaded into the target.

Additional advantages of the various embodiments of the invention willbe apparent to those skilled in the art upon review of the disclosureherein and the working examples below. It will be appreciated that thevarious embodiments described herein are not necessarily mutuallyexclusive unless otherwise indicated herein. For example, a featuredescribed or depicted in one embodiment may also be included in otherembodiments, but is not necessarily included. Thus, the presentinvention encompasses a variety of combinations and/or integrations ofthe specific embodiments described herein.

As used herein, the phrase “and/or,” when used in a list of two or moreitems, means that any one of the listed items can be employed by itselfor any combination of two or more of the listed items can be employed.For example, if a composition is described as containing or excludingcomponents A, B, and/or C, the composition can contain or exclude Aalone; B alone; C alone; A and B in combination; A and C in combination;B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certainparameters relating to various embodiments of the invention. It shouldbe understood that when numerical ranges are provided, such ranges areto be construed as providing literal support for claim limitations thatonly recite the lower value of the range as well as claim limitationsthat only recite the upper value of the range. For example, a disclosednumerical range of about 10 to about 100 provides literal support for aclaim reciting “greater than about 10” (with no upper bounds) and aclaim reciting “less than about 100” (with no lower bounds).

The devices, systems, and methods of the appended claims are not limitedin scope by the specific devices, systems, and methods described herein,which are intended as illustrations of a few aspects of the claims. Anydevices, systems, and methods that are functionally equivalent areintended to fall within the scope of the claims. Various modificationsof the devices, systems, and methods in addition to those shown anddescribed herein are intended to fall within the scope of the appendedclaims. Further, while only certain representative devices, systems, andmethod steps disclosed herein are specifically described, othercombinations of the devices, systems, and method steps also are intendedto fall within the scope of the appended claims, even if notspecifically recited. Thus, a combination of steps, elements,components, or constituents may be explicitly mentioned herein or less,however, other combinations of steps, elements, components, andconstituents are included, even though not explicitly stated.

The term “comprising” and variations thereof as used herein is usedsynonymously with the term “including” and variations thereof and areopen, non-limiting terms. Although the terms “comprising” and“including” have been used herein to describe various embodiments, theterms “consisting essentially of” and “consisting of” can be used inplace of “comprising” and “including” to provide for more specificembodiments of the invention and are also disclosed. Other than wherenoted, all numbers expressing geometries, dimensions, and so forth usedin the specification and claims are to be understood at the very least,and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, to be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

By way of non-limiting illustration, examples of certain embodiments ofthe present disclosure are given below.

EXAMPLES

The following examples set forth methods in accordance with theinvention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

Example 1: Microfluidic On-Demand Engineering of Exosomes Towards CancerImmunotherapy

Abstract: Extracellular nanovesicles (1 μm), particularly exosomes(30-150 nm), are emerging delivery system in mediating cellularcommunications, which have been observed for priming immune responses bypresenting parent cell signaling proteins or tumor antigens to immunecells. In this example, a streamlined microfluidic cell culture platformfor harvesting, antigenic modification, and photo-release of surfaceengineered exosomes directly in one workflow is provided. The PDMSmicrofluidic cell culture platform is replicated from a 3D-printed mold.By engineering antigenic peptides on exosome surface (e.g., gp-100,MART-1, MEGA-A3), the effective antigen presentation and T cellactivation can be achieved. This has been demonstrated by using theon-chip culture of human blood-derived leukocytes for engineeringsecreted exosomes in real-time with melanoma tumor peptides.gp100-specific CD8 T cells which were purified from the spleen of 2Pmel1 transgenic mice was tested. Significantly higher T-cell activationlevel (˜30%) induced by engineered exosomes was observed compared tonon-engineered exosomes. This microfluidic platform serves as anautomated and highly integrated cell culture device for rapid, andreal-time production of therapeutic exosomes that could advance cancerimmunotherapy.

Methods and Materials

3D Printing and microfluidic device fabrication: three pieces of moldsfor PDMS chip fabrication, including a base, wall, and top magnet holderwere provided. The mold was designed by using the SolidWorks® 2017 andprinted out by the 3D printer of Project 1200 from 3D Systems. Themultiple pieces had the finest structure in 50 μm, and with channelheight at 50 μm. The cell culture chamber was designed with 1000 μmdiameter, 500 μm height chamber. All molds were coated with Sportlinepalladium at the thickness of 20 nm. All three pieces were assembledusing the PDMS chip. The PDMS was filled with a height under 500 μm, sothe cell culture chamber left an open end for chamber plug. PDMS wascast by a 10:1 ratio with a linker reagent, and incubated at thetemperature of 40° C. for 6 hours. After the PDMS cured, it could bepeeled out easily. Chip inlets and outlet were punched by using 0.75 mmpuncher. Piranha treated glass and PDMS were both high-voltage plasmafor at least 30 seconds. The PDMS chips were then post-bond on the hotpad at the temperature of 40° C. for 5 mins. The chips were cleaned byDI water, and sterilized by autoclave (at 121° C. for 30 mins).

On-chip cell culture and exosome collection, engineering, and releasing:The cell cartridges (8 mm coverslip) were first cleaned with distilledwater, and air dried inside the bio-hood. Then, they were autoclaved at121° C. for 30 mins. The cartridges were set in a 24-well plate, and 500μL of 0.1 mg/mL poly-D-lysine hydrobromide (MP Biomedicals) was added toeach well, and incubated at the room temperature for 5 mins. 1 mL of MDwater was added to each well for 3 mins and repeated for two times toclean the cell cartridges, and then sit for air dried inside thebio-hood and stored for future use.

4 μL β2-microglobulin (Sigma-Aldrich) and 10 μL of each protein (gp100,MAGE-A3, and MART-1) were mixed with 186 μL 1×PBS to the modificationsolution at a final volume of 200 μL. The B-inlet was kept blocked andthe modification solution was pumped from A-inlet and the washing bufferfrom C-inlet through the chip at the volume flow rate of 1 μL/min for 10mins, and 0.1 μL/min for 10 mins, and static was set for another 10mins. A washing step was processed from both A-inlet and C-inlet at thevolume flow rate of 1 μL/min for 15 mins. The bottom side magnet wasremoved and the near UV turned on to treat the major chamber for 10mins. Another washing step from A-inlet and C-inlet was applied at thevolume flow rate of 1 μL/min for 20 mins, to collect the calved exosomefrom outlet about 20 μL.

Ultracentrifugation and exosomes staining: The collected 20 μL exosomeswere added to the ultracentrifuge tube and diluted to the final volumeof 1 mL for centrifugation (Thermo Scientific™ Sorvall™ MTX) under 1,500rcf for 30 mins. The supernatant was removed and transferred to a freshultracentrifuge tube. The mixture was then processed at the speed of100,000 rcf for 1 hour. Exosomes were stained by the PKH67 GreenFluorescent Cell Linker Midi Kit for General Cell Membrane Labeling(Sigma-Aldrich). The staining solution was prepared with 2 μL of PKH67and 1 mL of diluent C. Any remaining solution in the tube was discardedand 1 mL of Diluent C was added to re-suspend with gentle pipetting. Thestained solution to the ultracentrifuge tube, pipette mixed, and reactedat the room temperature for 3.5 mins. 2 mL of FBS (exosome depleted) wasadded to quench the free dye. 1.5 mL of 0.971 M sucrose solution wasadded for density gradient centrifugation. Another 6.5 mL of completemedia was added to raise the volume to 10 mL. The ultracentrifuge wasset at 100,000 rcf for 1 hour. The supernatant was discarded and the dyering washed carefully without reaching the center of the ring. Another 2mL of 1×PBS was added to re-suspend the pellet. The ultracentrifuge atthe speed of 100,000 rcf was ran for another 1 hour. The supernatant wassucked away, and another 100 μL of 1×PBS added to re-suspend the pellet.All steps were kept under sterile condition, and 1 μL ofPenicillin-Streptomycin (ATCC®, Catalog #30-2300, Lot #63525409) wasadded to the collected exosome, to inhibit and kill bacteria remainingin the solution. The collected exosomes were stored at 4° C. for lessthan 1 week and stored at −20° C. for up to one month.

Exosome uptake: THP-1 cells (ATCC®, TIB-202™) was cultured by usingATCC-formulated RPMI-1640 Medium (ATCC®, Catalog #30-2001, Lot#64331683) plus 10% exosome-depleted FBS for the completed media. Themonocytes cells were sub-cultured at the number of 8*105/mL, and byusing the alternative media changing method. The cells were used forexosomes up taking experiment at the density of 5*10⁵/mL. 200 uL of themonocytes cells were transferred to the 48-well plate with totally 11wells. 20 uL of normal exosome (NE) was added to 5 wells, also 20 μL ofengineered exosome (EE) to another 5 wells, and one well left as anegative control. Time intervals were set at 0 hours, 0.5 hours, 1 hour,2 hours, 3 hours, and 4 hours. At each time section, 100 uL of cellsuspension media was removed from the cytocentrifuge, at the speed of400 RPM for 4 mins. Glass slides were collected and 100 uL of FixativeSolution (ThermoFisher®, Catalog #R37814, Lot #17B285301) was added tocells' spot. The mixture incubated at room temperature for 18 mins, andthen the solution removed. 100 μL of 1×PBS buffer was added to thecells' spot, and left to set at room temperature for 3 mins. 1×PBSbuffer was removed and the cells' spot gently washed by the distilledwater. The slide was dried without droplet remains on the slide, and 50μL of 500 nM DAPI (ThermoFisher®, Catalog #D1306, Lot #1844202) appliedto the cells' spot, covered from light, and incubated at roomtemperature for 4 mins. The DAPI solution was then quickly removed andfollowed with a sufficient amount of 1×PBS buffer twice with 2 mins foreach time. The cells' spot was washed with distilled water, and brieflydried without droplet remain on the slide. One drop of ProLong™ GoldAntifade Mountant (ThermoFisher®, Ref #P10144, Lot1887458) was appliedand the slide covered with 25×25 #1.5 coverslip without any trappedbubble. The slide was stored at room temperature for 24 hours beforeimaging under a confocal microscope.

Results

3D-printing molded microfluidic cell culture device for on-inlineharvesting exosomes: A facile and low-cost approach for making aPDMS-based on-chip cell culture microfluidic device using a 3D-printedmold has been developed. The culture chip contains an on-chip cellculture chamber with 1 mm diameter and 0.5 mm height for on-chip growingcells and collecting exosomes derived from culture medium at downstream.The cell culture chamber is left open on top for applying a PDMS-made,finger-push plug for medium exchange and pushing the medium todownstream collection channels. The bottom of the cell culture chamberhas an outlet channel about 200 μm wide and 200 μm high (B-Inlet) forintroducing culture medium to mix with immunomagnetic isolation beads(A-Inlet). The C-Inlet is used to introduce washing buffer driven by asyringe pump. FIG. 4 demonstrates the mixing process through the A-Inletand B-Inlet and exit to exosome isolation channel (serpentine channel)under the observation of the fluorescence microscope using afluorescence dye solution. FIG. 5(b) records the immunomagnetic beadsmixing within the serpentine channel. Human blood-derived leucocyteswere cultured in the culture device with the morphology showing in FIG.5(c). Few red blood cells were still observed as a cup shape. Thesecreted exosomes were isolated, captured, and photo-released from theoutlet of the chip, and characterized by SEM imaging shown in FIG. 5(d).

A photo-cleavable linker was conjugated with bi-function of biotin andNHS chemistry on both ends. The biotin group anchors the photo-cleavablelinker to the surface of streptavidin immunomagnetic beads, and the NHSgroup conjugates the MHC-I peptide via the primary amine, as shown inFIG. 7A. The MHC class I molecules are heterodimers that consist of twopolypeptide chains, α, and β2-microglobulin. The two chains are linkednoncovalently via interaction of b2m and the α3 domain. The other twodomains α1 and α2 are folded to make up a groove for binding to 8-10amino acid peptides (MHC-I binding peptide). The MHC-I/peptide bindingcomplex will be displayed to cytotoxic T cells consequently fortriggering an immediate response from the immune system. Once the MHC-Ipositive exosomes are captured by tumor targeting antigenic (TTA)peptide and retained by immunomagnetic beads within the capture chamberwith the magnetic field, the antigenic loading buffer with saturated TTApeptides will be introduced via C-Inlet to completely bind and occupythe rest available MHC-I peptide binding sites. This antigenic surfaceengineering process can substantially enhance the loading amount of TTApeptides to captured MHC-I positive exosomes and boost the potency toactivate T-cells.

Further characterized was the binding strength between MHC-I peptidemodified photo-release immunomagnetic beads with MCH-I positivesexosomes labeled with fluorescence as shown in FIG. 7B. The MHC-Iantibody serves as the positive control to evaluate the binding strengthbetween tumor targeting antigen peptides and MHC-I positive exosomes.Because of the stronger binding strength between MHC-I/peptide complex,it has a higher potential to activate T cell anti-tumor responses.gp-100 was shown to have a stronger ability to form MHC-I/peptidecomplex and the binding strength is even stronger than MHC-I antibody(95% vs 84.8%).

The performance of on-demand photo-release was characterized in FIGS.8A-8E. With the comparison between positive control and negativecontrol, the fluorescent-labeled exosomes were captured and released bymeasuring fluorescence intensity from beads aggregates under an invertfluorescence microscope. The SEM imaging approach was used to confirmthe photo-release process. By comparing the SEM imaging of bead surfacebefore and after photocleavage, there were no identifiable exosomeparticles presented on the surface of beads, indicating the goodphoto-release performance. The UV exposure time was characterized aswell for reaching 98% photo-cleavage rate within 8-minute UV exposure.The size distribution of engineered exosomes and non-engineered exosomeswas evaluated, which showed an appropriate size range of exosomesbetween 50 nm-200 nm, confirming that engineered exosomes aremaintaining good integrity.

The side-effect of UV exposure on exosome molecular contents wasinvestigated, which shows non-detectable changes in terms of exosomalproteins, DNAs, and RNAs under 10-minute UV treatment (FIG. 13).

In order to evaluate the potency and integrity of engineered exosomesreleased from a microfluidic cell culture device via on-demandphoto-release, the exosomes from a chip outlet was harvested and labeledwith green fluorescence. gp-100 engineered exosomes and non-engineeredexosomes was incubated with dendritic monocytes for monitoring cellularuptake with a one-hour interval. The cells were then fixed and thenuclei were stained with DAPI. The green dots shown in FIG. 9A arelabeled exosomes, which are abundantly distributed around cellularnuclei. The cellular uptake begins within one hour and the uptake speedis much faster than the non-engineered exosomes. After 4 hours, it wasobserved that both engineered exosomes and non-engineered exosomes werecleared by the lysosome pathway. This observation indicated that gp-100engineered exosomes are much more active for dendritic monocytes uptake.The expression of Cytokine IFN-γ was monitored from incubating gp-100engineered exosome with dendritic monocytes using ELISA. Compared withthe incubation of non-engineered exosomes, the IFN-γ expression levelwas much higher for 48 hours after continuously monitoring, with anearly 2-fold increase. The gray dash line in the FIG. 9B indicates thepositive control using PWM protein as the stimulator. The dendriticcellular morphology upon stimulation was shown in FIG. 14. Compared withnegative control without stimulation, the both PWM protein and gp-100engineered exosomes gave significant influence on changing to roundfloating dendritic cells. The gp-100 engineered exosomes showed higherstimulation rate for Cytokine IFN-γ production than control PWM proteinstimulation.

Further investigated was the potency of gp-100 engineered exosome foractivating CD8+ T cells undergoing proliferation and cytolysis. It wasobserved that gp-100 engineered exosomes have the capacity to activatetransgenic T cells in the presence of activated dendritic cells. Thegp100-specific CD8 T cells were purified from the spleen of 2 Pmel1transgenic mice by magnetic cell sorting and labeled with Cell TraceViolet proliferation dye. The purified T cells were cultured alone (Tcells only) and mixed at a 3:1 ratio with naïve JAWS cells (an immaturedendritic cell line derived from a C57BL/6 mouse), T cells+JAWS cells,or JAWS cells that were activated for 48 hours with 200 ng/mL (Tcells+Activated JAWS cells). Engineered exosomes bearing the gp100peptide were added to the T cell cultures at increasing ratios ofexosomes: dendritic cells (25, 50 and 100). FIG. 10A. The cells andexosomes were co-cultured for 5 days and then CD8 T cells were analyzedby flow cytometry for Cell Trace Violet dilution as a measure ofproliferation. With T cell only condition as the negative control, itwas observed that the proliferation rate of CD8+ T cells cultured withgp-100 exosomes activated JAWS showed more than 30% increase, whichindicated that gp-100 engineered exosomes has strong potency to activateT cell cytolysis. FIG. 10B. The developed microfluidic on-demandantigenic surface engineering and photo-release of exosomes could be apowerful tool for developing an effective exosome-based vaccine anddelivery system for advancing Cancer Immunotherapy.

Immunogenic potency was also investigated for bovine respiratorysyncytial virus (BRSV). T cells and activated JAWS cells were incubatedwith increasing concentrations of BRSV antimicrobial peptide-engineeredexosomes (exosomes engineered with Peptide 4: M187-195 peptideNAITNAKII, SEQ ID NO:4). The immune-stimulation of CD8+ T cellproliferation is linearly responded to the dose of engineered exosomeswhich is more effective than using high dose peptide vaccines. The BRSVengineered exosomes have the capacity to activate BRSV M-specific Tcells in the presence of activated dendritic cells. C57BL/6 mice wereimmunized twice subcutaneously with 20 nM BRSV M187-196 adjuvanted inQuilA. At least 4 weeks after the final immunization, the animals wereeuthanized and spleens were collected. CD8+ T cells and CD11c+ splenicdendritic cells were isolated by magnetic cell separation. CD8+ T cellswere labeled with Cell Trace Violet proliferation dye. The purified Tcells were cultured alone (T cells only), or were mixed at a 3:1 ratiowith CD11c+ splenic DC (T cells+DC). DC cells were left unstimulated orwere treated with 200 ng/mL LPS to induce DC activation. Engineeredexosomes loaded with the BRSV peptide using the above-describedmicrofluidic platform were added to the T cell cultures at increasingratios of exosomes: dendritic cells (25, 50 and 100). Negative controlwells did not receive exosomes. Positive control wells were treated with1 nM or 5 nM pure M187-196 peptide. The cells and exosomes wereco-cultured for 5 days and then CD8 T cells were analyzed by flowcytometry for Cell Trace Violet dilution as a measure of proliferation.The results are shown in FIG. 11. All results support that the disclosedmethod for capture, antigenic loading, and photo-release can effectivelyproduce antimicrobial peptide engineered exosomes which lead tosuccessful activation of T cells with high potency.

Example 2: In Vivo Administration of Engineered Exosomes

The above immunogenic potency study used transgenic mice which wereinjected with engineered exosomes using proprietary disclosed method.The exosomes were engineered with gp-100 or BRSV M187-196 peptide on thesurface using the above-described streamlined/continuous microfluidicprocess, and suspended in the PBS buffer for in vivo Intraperitonealinjection through tail. We observed no injection site reactions oradverse responses (injection site swelling, irritation, etc.) from theinjected mice. Mice were observed twice per day for the first 72 hoursafter the injections and no adverse reactions were noted, indicatinggeneral in vivo safety of the engineered exosomes and relatedcompositions.

Example 3: Nanographene Fabricated Nano Pom Poms for Robust Preparationof Small Extracellular Vesicles Assisting Precision Cancer Diagnosis andTherapeutics

Extracellular vesicles (EVs), particularly exosomes, are emerging indeveloping liquid biopsy diagnosis of cancer, as well as the therapeuticdelivery. However, due to heterogeneous populations from diverse celltypes, obtaining pure extracellular vesicles (EVs) that are specific totheir cellular origin and molecular information is still extraordinarychallenging, which substantially hindered the clinical utility. Herein,we introduced a novel 3D-structured nanographene immunomagnetic beadwith unique Nano “pom poms” (aka NanoPoms) morphology for specificmarker-defined capture and release of intact small EV (sEV)subpopulations from nearly all types of biological fluids, includinghuman blood, urine, cow's milk, and cell culture medium, etc. Theconjugated photo-click chemistry on bead surface enables the release ofintact, captured sEVs on demand for ensuring substantially enhanceddiagnostic specificity and sensitivity employed in non-invasivediagnosis of bladder cancer, as demonstrated by multi-omic analysisusing the next generation sequencing (NGS) of somatic DNA mutations,miRNA profiles, as well as the Western blotting and global proteomicanalysis. The Nanopoms prepared sEVs also showed distinctive in vivobiodistribution patterns specific to their subtypes with good biologicalactivity. Such superior purity with improved specificity for pathogenicEV subpopulations will provide a precision approach critically needed indeveloping EV-based precision cancer diagnosis and therapeutics.

Developing the diagnostic and therapeutic utility of EVs is emerging,which promoted tremendous progress in cancer diagnosis, regenerativetissue repair, immunotherapy, drug delivery, and gene therapy. EVs areliving cell-secreted membrane vesicles in multiple subpopulations,including membrane shedding microvesicles (100 nm-1000 nm), endosomalmultivesicular body released exosomes (30 nm-150 nm), and apoptoticcellular fragment vesicles (≥1000 nm). Due to such large heterogeneityand significant size overlap between vesicle populations, the consensushas not yet emerged on precisely defining EV subtypes, such as endosomederived exosomes which is highly relevant to the disease pathogenesis.Assigning EVs to a particular biogenesis pathway remains extraordinarilychallenging. The generic term of EVs is recommended by complying with2018 guidelines from the International Society for ExtracellularVesicles (ISEV) proposed Minimal Information for Studies ofExtracellular Vesicles (“MISEV”). However, significant attention hasbeen focused on the exosome type small EVs (sEVs) and their molecularcomponents (e.g., proteins, DNAs, mRNA and miRNA), which implicates avariety of physiological functions and pathological disease states. sEVsecretion is exacerbated in tumor cells and enriched with a group oftumor markers, as evidenced by increased presence in plasma and ascitespatients in variable cancers. However, currently there is nostandardized purification method for obtaining pure sEV populations thatare specific to their cellular origin and molecular information yet. Thepurification methods that recover the highest amount of extracellularmaterials, no matter with the vesicle or non-vesicular molecules, aremainly the precipitation polymer kits and lengthyultracentrifugation-based (UC) approach. Such isolation approach isunable to differentiate the sEV populations from different cellularorigin or other EV subtypes (e.g., microvesicles and apoptotic bodies),as well as free proteins. This is a significant concern particularly forstudying the tumor cells derived circulating sEVs. The bulk measurementof a mixture of vesicle populations could potentially mask the essentialbiosignatures, which severely impairs the development of cancerdiagnosis and the investigation of pathological mechanism. Moreover, thecurrent existing isolation methods are rather in low efficiency (e.g.,UC isolation at ˜5-25%) and involves multiple time-consuming manualsteps which is not scalable, yet unable to separate exosome sEVs fromvirus. Therefore, methods for recovering exosome-type sEVs withoutnon-vesicular components are urgently needed, although the recoverycould be less than total EVs.

Immunomagnetic capture for isolating specific EV populations has beenwell accepted, due to the simple and straightforward protocols andspecificity to their significant molecular markers. However, currentexisting particle capture approaches are either in small quality withmanual protocols, or bound to solid surface/particles, and unable torelease intact, pure EV subpopulations. In this work, we introduce novelimmunomagnetic particles featured with the unique 3D hydrophilic,nanostructured graphene-sheet layers (Nano pom poms) which is capable ofon-demand photo release of intact EV subpopulations from captureparticles (FIG. 15A-E). Nano-graphene is an emerging interface toenhance biorecognition and bioseparation owing to its rich surfacechemistry, large surface area, and small feature sizes comparable toEVs. Conventionally, the non-covalently assembled nano-graphene coatingsuffers from the instability in buffer solutions over time. Our methodinterfaces Fe₃O₄/SiO₂ core-shell particles (˜1 μm) with graphenenanosheets via carboxamide covalent bonds, which leads to substantiallyimproved stability in the aqueous sample solution (FIG. 16). The pompoms-like graphene nanosheets aggregate on particle surface, whichproduces the unique 3D nano-scale cavities in between for affinitycapture of only nano-sized vesicles (FIG. 15A-E and FIG. 16). Mostimportantly, the conjugated photo-click chemistry on particle surfaceallows the release of intact, captured sEVs on demand, which furtherensures the specificity for harvesting marker-defined sEVsubpopulations. Herein, we demonstrated the substantially improvedspecificity for isolating tumor cell derived circulating sEVs frombladder cancer patient urines, without any additional purificationsteps.

The urological tumors make up approximately 25% of all human cancers,and their recurrence and progression rate are ˜50-70% which is higherthan other tumors. Thus, the most bladder cancer patients requirelifelong monitoring of recurrence, and demand heavily on non-invasivediagnostic or prognostic tools for long-term follow-up. Current goldstandard diagnostic procedures for bladder cancer are cystoscopy andurinary cytology which are invasive and low sensitivity to smallpapillary or Cis tumors, and also frequently cause side effects such asdysuria, hematuria, or urinary tract infection. Urine EVs have become avaluable and promising source of biomarkers for urological tumordetection. Additionally, the group of enriched biomarkers carried byurine EVs offers the unmatched possibility to integrate multi-omic dataanalysis for precisely defining the onset and progression of the bladdercancer disease. In this paper, we used our developed NanoPomsimmunomagnetic particles to specifically isolate sEVs from bladdercancer patient urines, and analyzed the EV DNA mutations, microRNAprofiles, as well as the protein biomarkers and proteomic profiles thatare relevant to urological tumors. The results showed much higherspecificity and sensitivity for detecting urological tumor biomarkersfrom NanoPoms isolated sEVs compared to other ultracentrifugation orbeads-based isolation approaches, which is highly desired for developingliquid biopsy analysis of bladder cancer. We also tested the NanoPomsprepared intact EVs for in vivo biodistribution and assessed theirbiological activity, which showed viable biological property in vivowith distinctive subpopulation specificity. The in vivo study using NanoPom Poms prepared sEVs supports the potential for developing as aprecision drug delivery carrier.

Results

NanoPoms immunomagnetic particles enable specific capture and on-demandrelease of intact sEV subpopulations. Compared to other EV isolationapproaches, immunomagnetic beads can specifically define EVsubpopulations based on their surface markers, in turn, lead to a betterpurity to exosome type sEVs, which is highly desirable to harvestdisease pathogenesis relevant EV subpopulations in cancer liquid biopsyand early detection. The synthesis route and unique 3D nano-scale pompoms structure were shown in FIG. 15A-B. Much larger hydrophilic surfaceareas are available with unique pom poms morphology for immobilizationof higher density of affinity capture entities (e.g., antibodies,aptamers, and affinity peptides), in contrast to commercial Dyna beads(FIG. 15B right panel vs. left panel). FIGS. 15C and E showed both TEMand SEM images on capturing nano-sized EVs from bladder cancer patienturines, which indicates the dense, round-shaped, nano-sized EVs (˜100nm) completely covering the particle surface and are completely releasedand separated from particles after photorelease. The X-Ray PhotoelectronSpectroscopy (XPS) analysis of surface chemistry, SEM imaging ofparticles before capture and after release, as well as the fluorescentbinding analysis were also performed to evaluate sEV capture performanceand capacity (FIG. 16). More biological fluids including cancer patientplasma, cell culture medium from mesenchymal stem cells, and cow milkswere also used to validate NanoPoms EV preparation approach as a genericand robust sEV isolation platform (FIG. 17 and FIG. 18). This operationprotocol is also much simple and cost-effective, amenable for scalingup, sterilization settings, and GMP operations (see Table s1).

The conjugated photo-click chemistry on the surface of NanoPomsimmunomagnetic particles enables the on-demand, light triggered releaseof captured sEVs (FIG. 15 and FIG. 16B). The released sEVs purified frombladder cancer patient urines were characterized using nanoparticletracking analysis (NTA) in comparison to UC isolation (FIG. 15E). Muchnarrower size range around 100 nm is observed, while UC isolated EVs arein the size range from 150 nm-400 nm. Although the total sEV number isless than UC isolated EVs, we hypothesize that NanoPoms particlesisolated sEVs could be more specific and purer to their cellular originand molecular information. In order to prove such isolation specificityand purity, we further characterized the exosomal molecular contentsusing the next generation sequencing (NGS) for identifying somatic DNAmutations and miRNA profiles, as well as the western blotting and globalproteomic analysis of tumor protein markers.

NGS Analysis of Tumor-Specific DNA Mutations Carried by Urinary EVs.

Bladder cancer (BC) is characterized by a large number of geneticalterations. EVs have been found carrying double-stranded DNA fragments,and genomic alterations in cancers. Thus, detecting DNA mutationscarried by urinary tumor EVs is emerging, yet challenging, due to theneeds of highly pure sample preparation which enables the sensitivedetection. The 11 BC patient urine samples were used to isolate EVs byUC, NanoPoms, and commercial bead approaches in parallel, with 4 healthyurine samples as the control group. The NGS GeneRead AIT panel was usedto identify the most cancer relevant 1,411 variants. UC preparation wasfound insensitive on cancer relevant variant detection, as it requiresmuch larger urine sample input (4 mL) with more than 100 ng EV DNAs togive detectable variant signals (FIG. 19A). We suspect that UC isolatedEV DNAs contain more genes which are not specific to cancer. The PDGFRAvariant (c.1432T>C, p.Ser478Pro) with 56.8% frequency was detected froma healthy individual in the control group using UC preparation, but notfrom NanoPoms preparation. In the BC disease group, NanoPoms isolatedsEV enabled much enhanced detection sensitivity and specificity to BCrelevant mutations including KRAS, PIK3CA, and ERBB2, which onlyconsumed 1 mL urine sample with using about 10-50 ng sEV DNAs. However,commercial bead isolated EVs using the same urine sample input did notyield sufficiently enriched DNAs for sensitive detection of cancerrelevant variants (FIG. 19A). In order to validate whether the genemutations found in urinary EVs are from the urological tumor, weevaluated the matched patient tumor tissue. The NGS GeneRead analysis oftumor tissue cells showed the consistent mutations of KRAS and ERBB2which also were presented in the urinary EVs from the same BC patient.Although as one might expect, more mutations were detected in the tumortissue, including MTOR and BRCA1; however, the pathogenic PDGFRA variant(c.1939A>G, p.Ile647Val) was found in the urinary EVs from both UC andNanoPoms preparations, but not in the tumor tissue cells (FIG. 19B). Itis worth mentioning that the PDGFRA variant (c.1939A>G, p.Ile647Val) hasbeen recognized as the tumor marker from the bladder urothelialcarcinoma and the gastrointestinal stromal tumor, which indicates thatcirculating sEVs could be a good surrogate and biomarker resources fortumor cells.

We also analyzed urinary EV-derived DNA mutations from both UC andNanoPoms preparations using ddPCR. A total of 30 bladder cancer patienturines were analyzed with 10 healthy individuals as control group. Withthe same EV DNA input (10 μg), EGFR (Thr790Met) and TERT (C228T andC250T) were detected. We observed much higher signal amplitudes fromNanoPoms prepared EV DNAs than that from UC approach (FIG. 19C and FIG.20). The average patients' EGFR Wt copy number is 3185.4±468.3 fromNanoPoms approach, which is 12.8-fold higher than that from UC approach(248.9±46.4) with 3-fold higher mutation detection efficiency (FIG. 20).The overall detection signal to base ratio from patient group isstatistically higher than that from control group (FIG. 19C) withsignificant diagnostic value (FIG. 19D) from NanoPoms preparation, incontrast to UC preparation which is unable to differentiate patientgroup from the healthy control group (p>0.05). This result indicates thehigh potential of NanoPoms prepared sEVs for developing more accurateliquid biopsy cancer diagnosis.

Interestingly, we also observed EGFR heterozygous mutations in three BCpatients while conducting ddPCR analysis of NanoPoms prepared urinarysEV DNAs (see FIG. 21). In contrast, UC isolates from the same patients2 and 3 could not show such heterozygous mutation (FIG. 22 and FIG. 21).In order to further validate this observation, we obtained the matchedpatient plasma and buffy coat with white blood cells (WBC) as thecontrol. NanoPoms preparation allows to pull out marker specific sEVpopulations based on the exosomal surface markers (CD9, CD63, and CD81)to match urinary EV populations, which avoids the interferences fromother microvesicles or non-pathogenic vesicles. Afterwards we usedSanger sequence to confirm the presence of the EGFR heterozygosity forpatients 1, 2 and 3. Results were consistent with ddPCR analysis fromNanoPoms preparation. As expected, the EGFR heterozygosity was notdetected from matched patient WBCs. Thus, these results clearly supportthat marker specific capture and release enabled by NanoPoms beads cansignificantly enrich tumor-associated sEVs for sensitive mutationdetection. Although the UC preparation yields larger numbers of vesicleparticles, their specificity and purity to tumor-associated sEVs aremuch less.

NGS Analysis of Urinary EV Small RNAs.

Analyzing RNAs within urinary EVs has been emerging with needs fornon-invasive, early detection, and timely medical checkup of BC. EV longnon-coding RNAs (lncRNAs) PVT-1, ANRIL and PCAT-1 have been reported asthe novel biomarker in BC diagnosis. However, NGS profiling of microRNAfrom tumor derived urinary EVs has not been exploited yet, whichrequires highly pure and consistent sample preparation. In this study,we used NanoPoms approach to purify sEVs from both BC and the healthyindividual urine samples for comparing urinary EV microRNA profiles withUC preparations. The distribution of small RNA categories from NanoPomsEV preparation showed more lncRNAs in both the BC group and healthycontrol group (42% from NanoPoms vs. 18.9% from UC) (Table s2). Incontrast, UC preparation leads to the higher percentage of tRNA.Although the exact role of EV lncRNAs is not well understood yet,several studies have showed exosomal lncRNAs are novel biomarkers incancer diagnosis and are highly associated with cancer progression andcellular functions. Only a small number of lncRNAs has been investigatedwhich may be due to the variation and uncertainty imposed by EVpreparation differences.

We further look into the top 100 miRNAs expression profiles as shown inFIG. 23B. The heatmap clustering analysis indicates the cleardifferentiation between BC group and healthy control from NanoPomspreparation, in contrast to UC preparation. We also investigated theinfluence of photo cleavage process on the integrity of overall EVmiRNAs (FIG. 24). We did not observe significant differences in heatmapprofiles with and without photo cleavage process. Generally, miRNAs arenot stable and sensitive to environmental changes. The NanoPoms EVpreparation is able to maintain the differentiation ability between BCand healthy control groups, in contrast to UC approach.

In order to further interpret urinary EV miRNA profiles and characterizethe influences imposed by EV sample preparation steps, we used thevolcano plot to analyze the statistical significance (P value) versusfold-gene expression changes from both UC and NanoPoms preparations. Itis interesting to note that top 10 miRNAs were highly enriched from theNanoPoms preparations, including hsa-miR-3168, hsa-miR-92b-5p,hsa-miR-891a-5p, hsa-miR-934, and hsa-miR-6785-5p (FIG. 23C and Tables3). We searched the reported miRNA functions and found those miRNAswere reported as the cancer relevant markers specifically sorted intoexosomes (Table s3). For instance, hsa-miR-3168 has been reported to beenriched in exosomes via a KRAS-dependent sorting mechanism incolorectal cancer cell lines and is known as the melanoma mature miRNA.The miR-92b-5p has been found to play a critical role in promoting EMTin bladder cancer migration. The hsa-miR-934 is an essential exosomaloncogene for promoting cancer metastasis. NanoPoms preparation offeredmuch higher molecular relevance with tumor derived exosomes, which iscrucial for developing biomarkers in liquid biopsy analysis of cancers.

Proteomic Analysis of Urinary EV Proteins.

By introducing the non-invasive urinary protein biomarkers, thecystoscopic evaluations in BC diagnosis can be confirmed with improvedaccuracy, which has significant clinical values. EDIL-3 (Epidermalgrowth factor (EGF)-like repeat and discoidin I-like domain-containingprotein 3) and mucin 4 (MUC 4) both have been found in exosomes purifiedfrom BC patient urines. We selected exosomal markers CD9, CD63, andTSG101, as well as the EDIL-3 and MUC4 for Western blotting analysis ofurinary EV proteins prepared by both UC and NanoPoms, with the humanbladder carcinoma cell line HTB9 as the control (FIG. 25A). The exosomalmarkers CD9, CD63, and TSG101 were consistently expressed in urinaryEVs, HTB9 cells and their EVs, which indicates consistent isolation ofEVs, although there is no significant difference between two preparationmethods. The expression level of EDIL-3 is significantly higher in BCpatients than healthy individuals, but not in the tumor cell line ortheir EVs from conditioned media. MUC4 protein marker was only observedin the human urinary EVs and HTB9 EVs, but not in HTB9 cells. Thisobservation supports the previous report that EDIL-3 and MUC 4 arehighly promising biomarkers in developing urinary EV-based BC diagnosisand prognosis tests.

The proteomic profiling of urinary EVs from both UC and NanoPomspreparations was shown in FIG. 25B. The identified proteins werecompared with the ExoCarta Exosome Protein Database and the UrinaryExosome Protein Database. Several proteins associated with exosomebiosynthesis were observed, such as proteins PIGQ and PAPD7 involved inGolgi apparatus, the cytosol protein S100-A7 and A9 found within theexosome lumen which is engaged with natural membrane budding processduring multivesicular body formation. We also observed a diverse groupof cytosolic enzymes (glyceraldehyde-3-phosphate dehydrogenase) andcytoskeletal constituents (actin, Beta-actin-like protein 2 ACTBL2, andmyosin-9). Although the majority of proteins are shared identificationswithin BC patient and healthy control groups (˜65%), as well as thedatabases we used, interestingly, we found 10 proteins which areuniquely identified only from BC patient using NanoPoms preparation(Table s4). Those proteins have previously been reported to beassociated with bladder cancer metastases, including IRAK4, KRT23, andRALGAPA2 (full list in Table s4). Also 4 proteins were found uniquely inthe healthy group using NanoPoms preparation, but not reported byExoCarta and Urinary Exosome Protein Databases. From the Human ProteinAtlas database (www.proteinatlas.org/), those proteins are intracellularand associated with vesicles, Golgi apparatus, and secreted pathway. Theidentifications are broadly consistent with that expected for exosomesand compatible with other researchers' investigations. Approximately 35%of proteins do not overlapped between the BC patient and the healthycontrol, which further support the utility of NanoPoms prepared sEVs foraiding the diagnosis of BC.

Identified proteins were classified by encoding genes which indicate themajority are located within membranous vesicles, cytosol, cytoplasm, andthe cytoskeleton, and some are located in Golgi (FIG. 25C). Thebiological processes associated proteome revealed significantassociations with regulation of biological process, metabolic process,response to stimulus, cell organization and biogenesis, transport, andthe cell death. The protein binding molecular function from thisproteome is dominant. Results exhibit good specificity to exosomalproteome, indicating NanoPoms preparation could provide a pure andhigh-quality exosome type sEVs, which facilitates the important researcharea in EV proteomics or multi-omics.

In Vivo Biodistribution Study of NanoPoms Prepared sEVs.

The NanoPoms preparation of sEVs via marker specific capture and releaseis able to collect intact, pure and homogenous sEV subtypes. Due to theon-demand, light-triggered release process, the molecular engineering,such as the surface modification, drug loading, or dye labeling, can beimplemented to immunomagnetically captured EVs before washing andreleasing. This protocol avoids the redundant post purification of smallmolecules from isolated sEVs, which is often challenging and causescontaminations. For instance, the remaining free dye during in vivotracking of EVs could cause false signals with longer distribution halftime, unspecific staining or tissue accumulation. With such uniquecapture and on-demand release, we can prepare pure and intact sEVsamples without contamination. In this study, we prepared sEVs from HTB9cells and non-malignant HEK cells with DiR labeling for intravenous tailinjection into BALB/cJ mice. The buffer solution from beads washing step(without EVs) was used as the negative control. From theserepresentative images in 24, 48, and 72 hr time intervals postinjection, the fluorescent whole mouse imaging is unable to provideenough precision to describe the levels of sEV distributions in tissueorgans (FIG. 26A). Thus, to minimize signal interference, the organswere harvested and imaged ex vivo in the time intervals of 48 and 72 hr,respectively (FIG. 26B, 26C). To rule out of the signal originating fromthe blood in the organs or from the free dye, we normalized the EVtracking signal with the negative control signal to affirm the in vivoEV tacking. In fact, the negative control images did not show muchdetectable signals indicating no remaining free dye during in vivotracking of sEVs as the background signal. By further observing theharvested organs, HTB9-derived sEVs exhibit different biodistributionprofile in lung, liver, kidney, spleen, heart, and brain, as compared tosEVs isolated from the non-malignant HEK293 cells. sEVs prepared fromthe HTB9 tumor cells were more concentrated in the liver and spleen withgradually increased intensity from 48 hrs to 72 hrs post injection. Incontrast, non-malignant HEK293-derived sEVs tend to spread from liver tolung and spleen after 48 hrs post injection. Although HTB9 sEVbiodistribution profile has not been reported elsewhere previously, theHEK293 sEV biodistribution profile is consistent with reported study inC57BL/6 mice. FIG. 26C provides the repetitive and quantitative analysisof biodistribution pattern over time. The results potentially indicatethe distinctive biodistribution profile from cancer-associated sEVswhich could be very important for understanding tumor cell-mediatedcommunications within the microenvironment. Currently, substantialefforts have been made for using sEVs as therapeutic agents or deliveryvehicle in vivo. Thus, being able to reproducibly prepare pure andhomogenous sEVs is critical for maintaining consistent biodistributionpatterns.

Discussion

All living cells secret EVs which are diverse populations withheterogeneous molecular functions. Recent and substantial research hasshown the heterogeneity of EVs in terms of density, molecular cargos,and morphology, which are even released by a single cell type. Ourrecent study also observed that molecular packaging of secreted EVs orexosomes is highly variable upon the change of cellular cultureenvironment as well as surrounding community. Thus, the more advancedanalytical methods are urgently needed to be able to decipher suchheterogeneity in precision. The bulk measurements could average out ormask essential disease associated signaling markers, leading to themisinterpretation of mechanisms. Additionally, for therapeutic delivery,the well-defined molecular components from the homogenous EV populationis also critical to precisely maintain controllable biodistributionpattern and delivery behavior. Due to the unique 3D nanographenestructure and specific marker defined capture-release process, ourdeveloped NanoPoms isolation approach focuses on the pure and homogenoussEV subpopulations for advancing the clinical utility.

The NGS analysis in our study demonstrated that DNAs isolated fromNanoPoms prepared sEVs are enriched for tumor-associated DNA mutationswhich are highly relevant to the bladder cancer (FIG. 19A) andcomparable to matched patient tumor tissue cells (FIG. 19B). The ddPCRanalysis also confirmed such performance with significantly higherdetectable copy numbers (FIG. 19C and FIGS. 20 and 21). Theheterozygosity also can be readily detected from very low level of copynumbers in NanoPoms prepared sEV DNAs, as confirmed by Sanger sequencingwith matched patient plasma and buffy coat (FIGS. 21 and 22A-B). The NGSanalysis of sEV RNAs prepared by NanoPoms also reveals the distinctiveprofiles between BC patient and healthy individual, in terms of RNAtypes and miRNA levels, in contrast to UC sample preparation (FIG.23A-B, Table s2). Most importantly, the top 10 miRNAs identified fromNanoPoms sEV preparation are highly relevant as the important cancermarkers specifically sorted into exosomes (FIG. 23C and Table s3). Thisevidence further supports that specific cancer-associated biomarker areenrich in exosome type circulating sEV and can serve as surrogates fortumor cells.

The miRNAs represent the most dynamic nucleic acid cargos in EVs, whichis relatively unstable and sensitive to external stimulus and changes.Thus, in order to gauge the impact of light release process on sEVisolation via NanoPoms approach, we compared the miRNA profiles with orwithout light release process, which did not show statisticallysignificant differences based on dendrogram clustering analysis (FIG.23B and FIG. 24). The light release process also is able to ensure thespecificity via releasing specifically captured sEVs only, notnon-specific binders. This data supports the quality and integrity ofNanoPoms prepared sEVs as a novel, rapid, and easy-to-use method.Currently, although urinary miRNA profiling is highly essential for BCdiagnosis, such study and relevant database have not been fullyestablished yet. NanoPoms based EV sample preparation could potentiallyspeed up this research direction by offering much simple and accuratesample preparation.

The urinary sEV cargos at the protein level from our study reveals theconsistent expression of exosomal proteins CD9, CD63, and TSG101 fromboth patient urinary EVs and cell lines from both UC and NanoPomspreparations. However, EDIL-3 levels have been observed much higher inBC patient urinary EVs compared to healthy individuals, which isconsistent with reported literature, indicating the high-qualitypreparation of exosome sEVs using NanoPoms approach (FIG. 25A). Further,the proteomic profiling also supports that NanoPoms prepared urinary sEVproteins can be used to differentiate BC disease from healthy status(FIG. 25B-C, and Table s4) with unique identification of pathogenic EVproteins, suggesting a promising avenue using NanoPoms prepared sEVs todevelop non-invasive bladder cancer diagnosis.

In order to further prove the integrity and biological activity ofNanoPoms prepared sEVs, in vivo biodistribution study exhibitsdistinctive distribution patterns between tumor-associated sEVs andnon-malignant sEVs (FIG. 26). This result may indicate that differentsubtypes and sources of EVs could have impact on the performance of drugdelivery while using EVs as the carrier. To date, the therapeuticpotential of different subpopulations of EVs, even from a single celltype, is not well known. It has been discussed that possibly only asmall fraction of the EVs from a cell can mediate the therapeuticeffects, and another EV population could have opposing effects. Thus,the reproducible isolation of specific sEV subpopulations are essentialto support the development of EV-based methods for effective therapeuticdelivery. However, current existing EV isolation strategies are stillunable to reproducibly differentiate sEV subpopulations. Our NanoPomsapproach for enriching exosome type sEVs with marker definition couldopen a new avenue for preparing pure and homogenous sEVs with improveddiagnostic and therapeutic efficacy.

Methods

NanoPoms immunomagnetic particles fabrication and characterization. Theproprietary bead fabrication follows the protocol of Fe₃O₄/SiO₂core-shell-based particle (˜1 μm) method with surface anchored grapheneoxide nanosheets via carboxamide covalent bonds and EDC/NHS chemistry,and further modified with (3-aminopropyl) triethoxysilane (APTES) andstreptavidin (Vector Laboratories, SA-5000). Beads were washed with PBSTthen resuspend in 1 ml PBST and 0.09% NaN₃ solution for storage at 4° C.In this study we used the pan capture with a mixture of CD9, CD63, andCD81 antibodies for bead-conjugation. For in vivo biodistribution study,we used CD9 antibody conjugated NanoPoms particles beads to prepare HTB9and HEK cells derived sEVs. After bead fabrication and conjugation, XPSanalysis was used (PHI 5000 VERSA PROBE II) with an Al anode of thex-ray source (46.95 eV) and 100μ X-ray beam size for operating at 23.2W. The power of the source was reduced to minimize X-ray damage foranalyzing EVs on bead surface.

The EV isolation from patients' plasma, urine, or cow milk andconditioned cell culture media were performed by incubation of 100 ulantibody-beads complex with 1 mL of samples at 4° C. overnight. Afterwashing, the photo release was performed using Analytikjena UVP 2UVTransilluminator Plus at 365 nm wavelength at 4° C. for 15 min (˜6mW/cm²). The UC isolation of EVs followed the well-documented protocolspublished previous. Briefly, to remove any possible apoptotic bodies andlarge cell debris, the supernatants were centrifuged at 10,000 g for 30mins, then transferred to ultracentrifuge tube (Thermo Scientific, USA)for ultracentrifugation at 100,000 g for 70 min (Sorvall™ MTX150Micro-Ultracentrifuge, USA), with second ultracentrifugation (100,000 gfor 70 min) for finally collecting EV pellets. The size characterizationof EVs was performed using the nanoparticle tracking analysis (NTA)Nano-Sight LM10 (Malvern Panalytical). Post-acquisition parameters wereadjusted to a screen gain of 10.0 and a detection threshold to 5.Standard 100 nm nanoparticles were used for calibration. Appropriatesample dilution in 1×PBS was evaluated before every measurement withfive repeats for each measurement.

sEV DNA extraction and NGS sequencing. Frozen urine samples were thawedovernight at 4° C. and pre-centrifuged at 4° C. 10,000 g for 30 min toremove cell debris. By using NanoPoms isolation, the extracted sEVs weretreated with DNase I before DNA extraction. The QIAamp DNA Mini Kit(Qiagen, 51304) was utilized to extract DNA from all EV samples. Theaddition of 1 μl of an aqueous solution containing 10 μg of carrier DNA(poly dA) to 200 μl Buffer AL was used to ensure binding conditions areoptimal for low copy number DNA according to the manufacturer'sprotocols. DNA was Eluted in 20 μL Buffer AE. DNA concentrations weremeasured using a Nanodrop platform at an absorbance at 260 and 280 nmsubtracted by the background value of carrier ploy dA only.

The library preparation by targeted enrichment using Qiagen GeneReadQIAact AIT DNA UMI and GeneRead clonal Amp Q Kits, was subjected tonext-generation sequencing (NGS) to generate FASTQ files (text-basedformat for storing nucleotide sequences). This test is a targeted NGSPanel that encompasses 30 genes and 1411 variants (AKT1, ALK1, BRAF,CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FBXW7, FGFR1, FGFR2,FGFR3, FLT3, GNA11, GNAQ, HRAS, KIT, KRAS, MAP2K1, MAP2K2, MET, NOTCH1,NRAS, PDGFRA, PIK3CA, RAF1, SMAD4, STK11) with variable full exon orpartial region. The reads are mapped to the Homo_sapiens_sequence hg19reference and variants identified using QIAGEN QCI-Analyze pipeline.

The extracted DNAs were amplified by PCR to detect the EGFR(P00533:p.Thr790Met) mutation using forward 5′-ATGCGTCTTCACCTGGAA-3′(SEQ ID NO:60) and reverse 5′-ATCCTGGCTCCTTATCTCC-3′ (SEQ ID NO:61)primers. Primers were designed by Primer3Plus online. The PCR assay wasperformed with Promega GoTaq Flexi DNA Polymerase kit in a 50-μL mixturecontaining 10 μL of 5×PCR buffer, 0.25 μL GoTaq Flexi DNA Polymerase, Mof each primer (IDT, USA) and 20 μL of DNA in an ABI PCR instrument(Applied Biosystems). The PCR conditions were as follows: Initialdenaturation at 95° C. for 2 min, followed by 35 cycles at 95° C. for 15sec, 54° C. for 30 sec and 72° C. for 40 sec, then a hold at 72° C. for5 min and a final permanent hold at 4° C. The 319 bp DNA size of PCRproducts were clarified by 1% agarose gel electrophoresis using 5 μL PCRproducts and remained DNA were purified by QIAquick PCR Purification Kit(Qiagen, 28104). The purified PCR products were sequenced by SangerSequencing approach (GeneWiz, USA) using the same primers above.

EV RNA extraction and NGS sequencing. The miRNeasy Mini Kit (Qiagen,217004) was used to extract total RNA from all EV samples permanufacture's protocols. The amount of 700 μL QIAzol lysis reagent wasadapted according to the manual. To achieve a higher RNA yield, thefirst eluate of 30 μL was applied to the membrane a second time.Isolated RNAs were quantified by High Sensitivity RNA ScreenTape Assayusing Agilent TapeStation 2200 (Agilent, 5067-5579, 5067-5580). TotalRNA was stored at −80° C. until small RNA Library preparation. TheQIAseq miRNA Library is prepared for Single Read 75 bp sequencing, withUMI tag per manufacture's protocols. After small RNA sequencing usingIllumina MiSeq system, the Qiagen specific UMI analysis per the kitinstruction was performed with details in supplementary information.

Droplet digital PCR. A pair of probes and a pair of primers weredesigned to detect EGFR and TERT mutation respectively. Due to the shortsize of the probe, in order to increase the hybridization properties andmelting temperature, Locked Nucleic Acid (LNA) bases were introduced onthe bases indicated with a “+”. One probe was designed to recognizewildtype (5′-TET/T+CATC+A+C+GC/ZEN/A+GCTC/-3′ IABkFQ, SEQ ID NO:62). Thesecond probe was designed to recognize the EGFR (P00533:p.Thr790Met)mutation loci, (5′-6FAM/T+CATC+A+T+GC/ZEN/A+GC+TC/-3′ IABkFQ, SEQ IDNO:63). Primers were designed to cover both side of detection loci. ForTERT, a probe was designed to detect both C228T and C250T mutation asboth mutations result in the same sequencing string, with (TERTMut:/56-FAM/CCC+C+T+T+CCGG/3IABkFQ/, SEQ ID NO:64). A second probe wasdesigned to recognize the C228 loci, also containing LNA bases, (TERTWT, /5HEX/CCCC+C+T+CCGG/3IABkFQ/, SEQ ID NO:65). Probes and primers werecustom synthesized by Integrated DNA Technologies (IDT). Amplificationswere performed in a 20 μL reaction containing 1×ddPCR Supermix forProbes (No dUTP), (Bio-Rad, 1863024), 250 nM of probes and 900 nM ofprimers and 8 μL EV DNA template. Droplets were generated using theQX200 AutoDG Droplet Digtal PCR System (Bio-Rad). Droplets weretransferred to a 96-well plate for PCR amplification in the QX200Droplet Reader. Amplifications were performed using the followingcycling conditions: 1 cycle of 95° C. for 10 minutes, then 40 cycles of94° C. for 30 seconds and 60° C. for 1 minute, followed by 1 cycle of98° C. for 10 minutes for enzyme deactivation. Keep all ramp rate at 2°C./sec. QuantaSoft analysis software (Bio-Rad) was used to acquire andanalyze data.

Western blot analysis. The 5 mL of each urine sample for two patientsand one healthy control were used for EV isolation and subsequentWestern blot analysis. 40 mls of HTB-9 conditional cell culture mediaand 40 mg cell pellets were also used as controls in this study. Sampleswere lysed in 1×RIPA buffer supplemented with protease inhibitors for 15min on ice. Only cell sample were ultrasonicated for 1 min. Proteinconcentration was quantified using Micro BCA Protein Assay Kit (ThermoFisher, 23235). The absorbances were read at 562 nm on a Synergy H1reader (BioTek). All sample concentration were adjusted to 0.1 μg/μL.Western blotting was performed under reducing conditions (RIPA buffer,β-mercaptoethanol and Halt Protease Inhibitor Cocktail, EDTA-Free) at95° C. for 5 min. 20 μL of protein lysate, each, were loaded onto 4-20%Mini-PROTEAN TGX Precast Protein Gels (BioRad, 4561093). The separatedproteins were transferred to a PVDF membrane (BioRad, 1620218). Afterblocking the membrane in Intercept (PBS) Blocking Buffer (LI-COR,927-70001) for one hour at room temperature, it was incubated over-nightwith the primary antibody at 4° C., followed by another incubation withthe secondary antibody for half hour at room temperature. The followingprimary antibodies were used, all diluted in blocking buffer (1:1000):anti-CD9 (Thermo Fisher, 10626D), anti-CD63 (Thermo Fisher, 10626D),anti-EDIL3 (Abcam, ab88667), anti-MUC4 (Abcam, ab60720), anti-TSG101(Invitrogen, PA5-86445), anti-ANXA7 (LSBio, LS-C387129-100). Thesecondary anti-mouse and anti-rabbit IRDye 800CW antibodies (LI-COR,926-32210 and 926-32211) were applied in 1:15,000 dilution. Imaging wereperformed by LI-COR Odyssey CLx system.

SEM and TEM. sEV-bead complex was resuspended in 200 μL cold PBSsolution. For electron microscope evaluation, EV-bead complexes werewashed with pure water followed by the fixation in a 2% EMS-qualityparaformaldehyde aqueous solution. 5 μL of sEV-bead mixtures were addedto cleaned silicon chips and immobilized after drying EVs under aventilation hood. Samples on silicon chips were mounted on a SEM stageby carbon paste. A coating of gold-palladium alloy was applied toimprove SEM image background. SEM was performed under low beam energies(7 kV) on Hitachi SU8230 filed emission scanning electron microscope.For TEM, ˜5 μL of each sEV-beads complex was left to adhere onto formvarcarbon coated copper Grid 200 mesh (Electron Microscopy Sciences) for 5mins followed by 5 mins of negative staining with 2% aqueous uranylacetate. Excess liquids were blotted by filter papers. Total gridpreparation was performed at room temperature till totally air-driedunder a ventilation hood for 25 mins. Images were acquired on the sameday at 75 kV using Hitachi H-8100 transmission electron microscope.

In vivo biodistribution analysis. The human bladder cancer cell lineHTB-9 (ATCC, 5637) and the negative control of human embryonic kidneyepithelial cell line HEK293 (ATCC, CRL-1573) were cultured in DMEM andMEM respectively, supplemented with 10% normal FBS and 1%penicillin/streptomycin. Once the cell cultures reached ˜70% confluency,the media was replaced with fresh media containing 10% exosome-depletedFBS (Thermo Fisher, A2720803). The cells were cultured for an additional72 h before the conditioned media were collected.

sEVs were isolated using Nanopoms approach. sEVs were incubated with 1mM fluorescent lipophilic tracer DiR(1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide)(Invitrogen, D12731) at room temperature (RT) for 15 minutes.DiR-labelled sEVs or free DiR dyes were segregated using Amicon Ultra-15Centrifugal Filter method. The 2.0×10⁹ particles/ml of isolated sEVsmeasured via NTA were used for each mouse injection. The 6- to8-week-old female BALB/cJ mice were used. The animal IACUC protocolshave been approved by the University of Kansas Institutional Animal Careand Use Committee with protocol number 258-01 and operated in the KUAnimal Care Unit. Freshly purified DiR-labelled sEVs were injectedthrough the tail vein for intravenous (i.v.) injection. The In-VivoSystems (Bruker, USA) with high-sensitive CCD camera was used forcollecting fluorescence, luminescence and X-ray images. Isofluranesedated live mice were taken fluorescence and X-ray images prior to theanimals were sacrificed, then main organs (brain, heart, lung, liver,kidney and spleen) were harvested for fluorescence imaging in 3 mins(excitation 730 nm, emission 790 nm), X-ray imaging (120 mm FOV, 1 min)and luminescence imaging (90 fov, 0.2 sec) at 24 h, 48 h and 72 h timepoints, respectively. The data were analyzed using the Bruker MIsoftware.

Supplemental Materials

Nanographene Fabricated Nano Pom Poms for Robust Preparation of SmallExtracellular Vesicles Assisting Precision Cancer Diagnosis andTherapeutics

Characterization of Nanopoms Immunomagnetic Particles for SpecificCapture and Release of sEVs from a Variety of Biological Fluids.

As shown in FIG. 18, the optimization of NanoPoms particlesconcentration used for isolating sEVs from 1 mL milk solution,characterized by the Pierce BCA Protein Assay. The five repetitivemeasurements were performed for each data point with RSD<˜5% (n=5). Withincreasing the amount of capture beads in 1 mL biofluids, more EVs wereisolated to gradually reach to the maximum. Once the available capturebinding sites on the bead surface excess the number of overall EVs, theincrease of beads amount does not influence on the overall EV captureamount. Thus, the 1 mg/mL of NanoPoms capture particles were used forappropriate capture efficiency ranging from ˜10⁸ to ˜10¹³ particles/mL.

TABLE s1 Comparative analysis of cost, time, steps, and performancebetween EV isolation methods. EV Isolation Chromatographic PolymerMethods NanoPoms Ultracentrifugation Column Precipitation Cost Low, noHigh, very Low Low instrument expensive required equipment cost Protocol~4 hrs ~12 hrs ~6 hrs ~6 hrs duration Processing From ~μL to L ~12-500mL ~150 μL-100 mL ~≤1 mL capacity (IZON) ExoQuick ™ Subtype Surfacemarker No No No specificity defined specificity to subtypes Purity Highto pathogenic Mixture of EV Mixture of EV Mixture of EV EVs populationspopulations with protein aggregates Reproducibility High Low — —Scalability Yes No Yes NoNanoPoms Prepared sEVs for Droplet Digital PCR Analysis withSubstantially Improved Detection Specificity and Sensitivity.

As shown in FIG. 20, both EGFR (Thr790Met) and TERT (C228T and C250T)mutations were detected, but the detection efficiency was 3-fold higherfrom NanoPoms isolated sEV DNAs as compared to UC approach. As shown inFIG. 21, the ddPCR analysis of NanoPoms isolated sEV DNAs showed theEGFR heterozygosity in the three bladder cancer patients. In contrast,UC isolated EV DNAs from the same sample input did not detect the EGFRheterozygosity in patient 2 and patient 3. The DNA copy number wassubstantially lower in UC prepared EV DNAs as compared to NanoPoms.

3. NGS Analysis of Urinary EV Small RNAs

Bioinformatics Analysis: The sequences of precursor miRNA, tRNA, snRNA,snoRNA, scaRNA, rRNA, scRNA, vaultRNA, lncRNA, miscRNA, pseudogenes,retained introns, ribozymes and transcribed unprocessed pseudogenes wereextracted from RefSeq (hg38) and GENCODE (v29) to build a customizeddatabase. We refer to this database as the customized ncRNA database.For further quantifying mature miRNA abundance, their sequences wereextracted from miRBase (ver. hg38) to form another database, which isrefer to as the mature miRNA database. Initial quality assessment of thereads was performed using the FASTQC(www.bioinformatics.babraham.ac.uk/projects/fastqc/) package. Cutadaptwas used to trim the 5′ (GUUCAGAGUUCUACAGUCCGACGAUC, SEQ ID NO:66) and3′ (AACTGTAGGCACCATCAAT, SEQ ID NO:67) adaptor sequences from thesequences. The unique molecular indices (UMI) sequences in the QIAseqlibraries were further trimmed using FASTP(--umi_loc=read1--umi_len=12). The trimmed reads were filtered with aminimum length of 15 nt. Mapping to the mature miRNA database andcustomized non-ncRNA database was performed using BWA (bwa-aln). Werelaxed the BWA parameter to allow each read to mapped to at most 100locations, accounting for the numerous multi-mapping cases induced bythe short-read length of the sequencing reads. To ensure maximumaccuracy, we further restrict perfect sequence in seeding (by setting−k=0). All other parameters were used as default. With an in-housescript, we counted the read mapping to quantify the expression level ofeach ncRNA gene and the abundance of each mature miRNA. Formulti-mapping, we evenly distributed its abundances to all mappedlocations. The mapping results against the customized ncRNA databasewere used to quantify different types of ncRNAs as the Pie charts (referpie-chart figures here). The mapping results against the mature miRNAdatabase were summarized as the volcano plot (refer volcano plot here).The mapping results were further analyzed using the Deseq2 to revealsignificantly different abundant miRNAs. The most significant 100 miRNAswere further selected to generate the heatmap (shown below).

TABLE s2 The distribution of small RNAs from urinary EV isolated by UCand NanoPoms. % tRNA IncRNA miRNA snRNA snoRNA miscRNA rRNA scaRNA UC-BCUrine EVs 64.35 18.89 12.40 1.92 0.18 0.50 1.39 0.01 Nano-Wing-BC UrineEVs 47.52 42.02 4.94 1.41 0.29 0.89 1.98 0.07 UC-Healthy Urine EVs 69.8814.68 8.59 4.66 0.23 0.46 1.09 0.01 Nano-Wing-Healthy Urine EVs 49.6242.10 4.19 1.16 0.38 0.51 0.85 0.01 % Mt-tRNA Mt_rRNA scRNA vaultRNAsRNA Other UC-BC Urine EVs 0.02 0.002 0.00 0.004 0.001 0.34 Nano-Wing-BCUrine EVs 0.03 0.01 0.00 0.002 0.001 0.82 UC-Healthy Urine EVs 0.0030.003 0.00 0.003 0.001 0.38 Nano-Wing-Healthy Urine EVs 0.03 0.01 0.000.002 0.001 1.14

TABLE s3 The top 10 highly enriched miRNAs identified from NanoPomsisolated urinary EVs as compared with UC. Genes Log2 Fold Change p valueReported signaling pathway/functions hsa-miR-3168 5.140358266 1.38E−07KRAS-dependent sorting to exosomes from colorectal cancer cell linesKnown Melanoma mature miRNA hsa-miR-92b-5p 4.358349687 1.08E−06 Cancermetastasis Promote EMT in bladder cancer migration hsa-miR-891a-5p−5.329381923 1.19E−06 Prognostic marker for HR-positive breast cancerhsa-miR-6785-5p 5.063745282 1.80E−06 miRNA target genes in the TP53signaling pathway in tumor hsa-miR-934 −4.078662275 3.10E−06 Cancermetastasis, exosomal oncogene Non-coding RNA with neurogenic functionhsa-miR-6883-3p 4.420682437 5.64E−06 Target CDK4/6 in colon cancer cellshsa-miR-3202 5.039882582 8.31E−06 Regulating TLR signaling pathwayPromoted Cell Apoptosis hsa-miR-3648 −4.682609314 1.30E−05 Promoteinvasion and metastasis of human bladder cancer Regulate cellproliferation hsa-miR-6802-5p 4.168335059 1.84E−05 Exosomal regulatorymiRANs Heart disease hsa-miR-6763-5p 4.0778099 2.21E−05 ImmunityRegulation

As shown in FIG. 24, the disease group can be significantlydifferentiated from healthy group regardless if the light releaseprocess was implemented.

4. Proteomic Analysis of Urinary EV Proteins

Urinary EV pellets resultant from ˜2 mL of urine from both bladdercancer patients and healthy individuals were reconstituted in 400 μL ofM-PER Mammalian Protein Extraction Buffer (Thermo) supplemented with 1×Halt Protease Inhibitors (Thermo) and sonicated in an ultrasonic waterbath for 15 min. Lysates were exchanged into ˜40 μL of 100 mMtriethylammonium bicarbonate using Amicon Ultra-0.5, 3 k columns(Millipore). Lysate were digested overnight with Trypsin Gold, MassSpectrometry Grade (Promega). Peptides were finally reconstituted into0.1% formic acid to a concentration of 0.1 μg/μL and injected into a1260 Infinity nHPLC (Agilent) with separation from a Jupiter C-18column, 300 Å, 5 μm, Phenomenex) in line with a LTQ XL ion trap massspectrometer equipped with a nano-electrospray source (Thermo). Allfragmentation data were collected in CID mode. The nHPLC was configuredwith binary mobile phases that included 10 min at 5% of 0.1% formic acidand 85% acetonitrile, 180 min (LTQ XL), 5 min wash using 70% of 0.1%formic acid, 85% acetonitrile, and 10 min equilibrate. Samples wereperformed in duplicate for obtaining the average values utilized foranalysis. Searches were performed with UniRef100 database which includescommon contaminants from digestion enzymes and human keratins. Peptideswere filtered and quantified using ProteoIQ (Premierbiosoft, Palo Alto,Calif.).

TABLE S4 The 10 unique gene products identified from BC patient only and4 unique genes identified from healthy control group only, by proteomicanalysis of NanoPoms isolated urinary EV proteins. The Human ProteinAtlas database was used: www.proteinatlas.org/ BC Patient FASTA TitleLines Reported Functions and Pathways ARMCX4 |Q5H9R4|ARMX4_HUMANIntracellular, Nucleoplasm and additionally in Vesicles Armadillorepeat-containing Prognostic marker, novel passenger cancer genesX-linked protein 4 DSC3 |Q14574|DSC3_HUMAN Plasma membrane, CellJunctions Desmocollin-3 Regulated by p53 signaling pathway in colorectalcancer Down-regulated in primary breast tumors IRAK4 |Q9NWZ3|IRAK4_HUMANIntracellular, Microtubules and additionally in Nucleoli, CytosolInterleukin-1 receptor- Prognostic marker in endometrial cancer andurothelial cancer associated kinase 4 Disrupts inflammatory pathways anddelays tumor development KRT23 |Q9C075|K1C23_HUMAN Intracellular,Intermediate filaments and additionally in Cytosol Keratin, type Icytoskeletal 23 Prognostic marker in urothelial cancer Keratin 23promotes telomerase reverse transcriptase expression and humancolorectal cancer growth PIGQ |Q9BRB3|PIGQ_HUMAN Golgi apparatus,Vesicles and additionally in Nucleoplasm Phosphatidylinositol N-Prognostic marker in renal cancer acetylglucosaminyltransferase GPI-APbiosynthesis deficiency disorder syndrome subunit Q SERPINB2|P05120|PAI2_HUMAN Intracellular Plasminogen activator Prognostic markerin urothelial cancer inhibitor 2 SerpinB2 inhibits migration andpromotes a resolution phase signature in large peritoneal macrophagesPDHA2 |P29803|ODPAT_HUMAN Intracellular, Mitochondria, links theglycolytic pathway to the tricarboxylic cycle Pyruvate dehydrogenase E1Testis specific component subunit alpha, In Tumor Suppressor genedatabase https://bioinfo.uth.edu/TSGene/, testis-specific form,mitochondrial RALGAPA2 |Q2PPJ7|RGPA2_HUMAN Intracellular, Plasmamembrane, Cytosol Rai GTPase-activating Prognostic marker in renalcancer protein subunit alpha-2 Downregulation of Ral GTPase-activatingprotein promotes tumor invasion and metastasis of bladder cancer SMARCD3|Q6STE5|SMRD3_HUMAN Intracellular, Nucleoplasm SWI/SNF-related matrix-Prognostic marker in colorectal cancer associated actin-dependent Thechromatin remodeler SMARCD3 regulates cell cycle progression and itsregulator of chromatin expression predicts survival outcome in ER+breast cancer subfamily D member 3 PAPD7 |Q5XG87|PAPD7_HUMANIntracellular, Nucleoplasm and additionally in Nuclear membrane, Golgiapparatus Non-canonical poly(A) RNA Prognostic marker in renal cancerand urothelial cancer polymerase Healthy FASTA Title Lines ReportedFunctions and Pathways ORM2 |P19652|A1AG2_HUMAN Intracellular, Vesiclesand additionally in Golgi apparatus Alpha-1-acid glycoprotein 2 Orm1 andOrm2 are conserved endoplasmic reticulum membrane proteins regulatinglipid homeostasis and protein quality control ATP5F1A |P25705|ATPA_HUMANATP Intracellular, Mitochondria synthase subunit alpha, Reduced Levelsof ATP Synthase Subunit ATP5F1A Correlate with Earlier-Onsetmitochondrial Prostate Cancer DEFB1 |P60022|DEFB1_HUMAN Secreted pathwayBeta-defensin 1 Normal tissue annotation MPP7 |Q5T2T1|MPP7_HUMANIntracellular, Cell Junctions and additionally in Nucleoplasm MAGUK p55subfamily Acts as an important adapter that promotes epithelial cellpolarity and tight junction member 7 formation via its interaction withDLG1. Involved in the assembly of protein complexes at sites ofcell-cell contact

The invention claimed is:
 1. A method for capture and photorelease ofextracellular vesicles from a test sample suspected of containing one ormore target extracellular vesicles, comprising: contacting said testsample with a plurality of immunomagnetic particles in a test containerto create a first assay mixture, said immunomagnetic particles eachcomprising: a central magnetic particle comprising a core particlehaving a surface; a graphene-oxide-layer coating said core particle,said graphene-oxide layer comprising graphene-oxide nanosheets, saidgraphene-oxide nanosheets being covalently bonded to the particlesurface; and at least one polydopamine polymer coupled with thegraphene-oxide nanosheets, a targeting moiety coupled to the at leastone polydopamine polymer, and a photocleavable linker, saidphotocleavable linker connecting said magnetic particle and saidtargeting moiety, wherein said target extracellular vesicles, ifpresent, are bound by said targeting moiety to create an immunomagneticcomplex; magnetically immobilizing said immunomagnetic complexes at afirst location in said container; washing said first assay mixture andresuspending said magnetically immobilized immunomagnetic complexes insolution to create a second assay mixture; exposing said immunomagneticcomplexes to activating radiation to cleave said photocleavable linker,wherein said target extracellular vesicles are released from saidimmunomagnetic complex to yield released extracellular vesicles andreleased immunomagnetic particles; magnetically immobilizing saidreleased immunomagnetic particles at a second location in saidcontainer, wherein said released target extracellular vesicles remainsuspended in the second assay mixture; and isolating said releasedtarget extracellular vesicles from the second assay mixture, andoptionally separately recovering said released immunomagnetic particles.2. The method of claim 1, wherein said test sample is selected from abiological fluid selected from the group consisting of cell culturemedium, tissue fluid, urine, milk, saliva, serum, plasma, blood,cerebrospinal fluid, nasal secretions, exhaled breath condensate, tears,adipose tissue, seminal fluid, vaginal secretions, synovial fluid,pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid,amniotic fluid, otic fluid, gastric fluid, placental fluid, breast milk,Perilymph fluid, ascitic fluid, and combinations thereof.
 3. The methodof claim 1, wherein said first or second assay mixture comprises wateror buffer system selected from the biological buffer consisting of, butnot limited to, phosphate buffered saline, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS, Bis-Tris,MOPSO, ADA, ACES, PIPES, BES, TES, MOBS, and mixtures thereof.
 4. Themethod of claim 1, wherein said assay volume ranges from 1 μL to 10 L.5. The method of claim 1, wherein said targeting moiety is selected fromthe group consisting of antibodies, aptamers, antigenic peptides, tissuetargeting/penetration peptides, MEW binding peptides, neoantigens, phagedisplayed epitopes, nucleic acid oligos, enzyme, receptors, inhibitors,and combinations thereof.
 6. The method of claim 1, wherein thephotocleavable linkage comprises biotin.
 7. The method of claim 1,wherein said test container is selected from the group consisting ofmicrofluidics chambers, test tubes, centrifuge tubes, microtubes,beakers, vials, flasks, bottles, ELISA well plate, 6-well, 12-well,24-well, 48-well, 96-well, or 384-well plates, and cell culturecontainers.
 8. The method of claim 1, wherein said immunomagneticparticles have a diameter of 5 nm or greater.
 9. The method of claim 1,further comprising detecting one or more biomarkers, or multi-omicanalysis in said released target extracellular vesicles.
 10. The methodof claim 9, further comprising rendering a diagnosis based upon said oneor more biomarkers.
 11. The method of claim 10, wherein said diagnosisis a cancer diagnosis, wherein said method is repeated over time, saidmethod further comprising monitoring the progression of said cancer overtime.
 12. The method of claim 10, wherein said diagnosis is a cancerdiagnosis, wherein said method is repeated after treatment for saiddiagnosis, said method further comprising monitoring the effect saidtreatment of said cancer.
 13. The method of claim 9, said assay havinghigh sensitivity for said one or more biomarkers.
 14. The method ofclaim 9, wherein said biomarkers are selected from the group consistingof nucleic acids, proteins, peptides, enzymes, inhibitors, lipids,metabolites, cytokines, stimulating factors, hormones, fragmentsthereof, and mutated forms thereof.
 15. The method of claim 10, whereinsaid diagnosis is a specific genetic mutation, or expression levelchanges of a biomarker or downstream product thereof, such as mRNA,miRNA, proteins, and peptides.
 16. The method of claim 1, wherein saidreleased target extracellular vesicles comprise a surface modified withone or more of said targeting moieties, said targeting moietiesremaining bound to said target extracellular vesicles after saidrelease.
 17. The method of claim 16, wherein said targeting moieties areactive agents.
 18. The method of claim 1, wherein said container is amicrofluidic mixing channel, wherein said magnetically immobilizing saidimmunomagnetic complexes at a first location comprises applying amagnetic field within a microfluidic chamber, and wherein afterphotolytically releasing said immunomagnetic particles and said targetextracellular vesicles, said magnetically immobilizing said releasedimmunomagnetic particles at a second location comprises immobilizingsaid released immunomagnetic particles in a microfluidic chamber,wherein said released target extracellular vesicles are washeddownstream to an outlet of the microfluidic mixing channel.
 19. Acomposition comprising a plurality of released target extracellularvesicles prepared according to claim 1, wherein said released targetextracellular vesicles each comprise a surface modified with one or moreof said targeting moieties, said targeting moieties remaining bound tosaid target extracellular vesicles after said release, wherein saidtarget extracellular vesicles remain intact after said release, whereinsaid targeting moieties are active agents.
 20. The composition of claim19, wherein said active agents are therapeutic or diagnostic nucleicacids, drugs, small molecule compounds, chemotherapeutics, stimulatingfactors, binding peptides, and/or antigens.
 21. A method of treatment ordiagnosis comprising administering to a subject in need thereof, acomposition according to claim
 19. 22. The method of claim 21, whereinsaid targeting moieties are released from said target extracellularvesicles before administering said target extracellular vesicles to saidsubject.
 23. A composition comprising a plurality of released targetextracellular vesicles prepared according to claim 1, wherein saidreleased target extracellular vesicles each comprise a surface modifiedwith one or more of said targeting moieties, said targeting moietieshaving been released from said target extracellular vesicles, whereinsaid target extracellular vesicles remain intact after said release.